Tumor necrosis factor receptor-associated factors

ABSTRACT

The invention concerns new tumor necrosis factor receptor associated factors, designated TRAF. The new factors are capable of specific association with the intracellular domain of the type 2 TNF receptor (TNF-R2), and are involved in the mediation of TNF biological activities,

FIELD OF THE INVENTION

The present invention concerns novel polypeptide factors. Moreparticularly, the invention concerns factors associated with the type 2tumor necrosis factor receptor (TNF-R2).

BACKGROUND OF THE INVENTION

Tumor necrosis factor (TNF, also referred to as TNF-α) is a potentcytokine produced mainly by activated macrophages and a few other celltypes. The large number of biological effects elicited by TNF includehemorrhagic necrosis of transplanted tumors, cytotoxicity, a role inendotoxin shock, inflammatory, immunoregulatory, proliferative, andantiviral responses reviewed in Goeddel, D. V. et al., Cold SpringHarbor Symposia on Quantitative Biology 51, 597-609 (1986); Beutler, B.and Cerami, A., Ann. Rev. Biochem. 57, 505-518 (1988); Old, L. J., Sci.Am. 258(5), 59-75 (1988); Fiers, W. FEBS Lett. 285(2), 199-212 (1991)!.The literature has reported that TNF and other cytokines such as IL-1may protect against the deleterious effects of ionizing radiationproduced during the course of radiotherapy, such as denaturation ofenzymes, lipid peroxidation, and DNA damage (Neta et al, J. Immunol.136(7): 2483, (1987); Neta et al., Fed. Proc. 46:1200 (abstract),(1987); Urbaschek et al., Lymphokine Res. 6:179 (1987); U.S. Pat. No.4,861,587; Neta et al, J. Immunol. 140: 108 (1988)!. A related molecule,lymphotoxin (LT, also referred to as TNF-β), that is produced byactivated lymphocytes shows a similar but not identical spectrum ofbiological activities as TNF (see, e.g. Goeddel, D. V. et al., supra,and Fiers, W., supra). TNF was described by Pennica et al, Nature 312,721 (1984); LT was described by Gray et al., Nature 312, 724 (1984).

The first step in the induction of the various cellular responsesmediated by TNF or LT is their binding to specific cell surfacereceptors. Two distinct TNF receptors of approximately 55-kDa (TNF-R1)and 75-kDa (TNF-R2) have been identified Hohmann, H. P. et al, J. Biol.Chem. 264, 14927-14934 (1989); Brockhaus, M. et al, Proc. Natl. Acad.Sci. USA 87, 3127-3131 (1990)!, and human and mouse cDNAs correspondingto both receptor types have been isolated and characterized Loetscher,H. et al, Cell 61,351 (1990); Schall, T. J. et al, Cell 61,361 (1990);Smith, C. A. et al., Science 248, 1019 (1990); Lewis, M. et al, Proc.Natl. Acad. Sci. USA 88, 2830-2834 (1991); Goodwin, R. G. et al., Mol.Cell. Biol. 11, 3020-3026 (1991)!. Both TNF-Rs share the typicalstructure of cell surface receptors including extracellular,transmembrane and intracellular regions. The extracellular portions ofboth receptors are found naturally also as soluble TNF-binding proteinsNophar, Y. et al, EMBO J. 9, 3269 (1990); and Kohno, T. et al., Proc.Natl. Acad. Sci. U.S.A. 87, 8331 (1990)!!. The amino acid sequence ofhuman TNF-R1 and the underlying nucleotide sequence are disclosed in EP417,563 (published 20 Mar. 1991), whereas EP 418,014 (published 20 Mar.1991) discloses the amino acid and nucleotide sequences of human TNF-R2.

Although not yet systematically investigated, the majority of cell typesand tissues appear to express both TNF receptors.

The individual roles of the two TNF receptors, and particularly those ofTNF-R2, in cell signaling are far from entirely understood, althoughstudies performed by poly- and monoclonal antibodies (mAbs) that arespecific for either TNF-R1 or TNF-R2 have provided some very valuableinsight into the functions and interactions of these receptors.

It has been observed that both polyclonal and monoclonal antibodiesdirected against TNF-R1 can act as specific agonists for this receptorand elicit several TNF activities such as cytotoxicity, fibroblastproliferation, resistance to chlamydiae, and synthesis of prostaglandinE₂ Engelmann, H. et al, J. Biol. Chem. 265, 14497-14504 (1990); Espevik,T. et al, J. Exp. Med. 171,415-426 (1990); Shalaby, M. R. et al., J.Exp. Med. 172, 1517-1520 (1990)!. Agonist antibodies to TNF-R1 withantiviral activity are disclosed in copending application Ser. No.07/856,989 filed 24 Mar. 1992.

In addition, polyclonal antibodies to both murine TNF-R1 and TNF-R2 havebeen developed, have been shown to behave as specific receptor agonistsand induce a subset of murine TNF activities. While the murine TNF-R1was shown to be responsible for signaling cytotoxicity and the inductionof several genes, the murine TNF-R2 was shown to be capable of signalingproliferation of primary thymocytes and a cytotoxic T cell line, CT6Tartaglia, L. A. et al, Proc. Natl. Acad. Sci. USA 88, 9292-9296(1991)!. The ability of TNF-R2 to stimulate human thymocyteproliferation has been demonstrated in experiments with monoclonalantibodies directed against the human receptor.

Monoclonal antibodies against human TNF-R1 that block the binding of TNFto TNF-R1 and antagonize several of the TNF effects have also beendescribed Espevik, T. et al, Supra; Shalaby, M. R. et al, Supra; Naume,B. et al, J. Immunol. 146, 3035-3048 (1991)!.

In addition, several reports described monoclonal antibodies directedagainst TNF-R2 that can partially antagonize the same TNF responses(such as cytotoxicity and activation of NF-KB) that are induced byTNF-R1 agonists Shalaby, M. R. et al, Supra; Naume, B. et al, Supra; andHohmann, H. P. et al, J. Biol. Chem. 265, 22409-22417 (1990)!.

It is now well established that although the two human TNF receptors areboth active in signal transduction, they are able to mediate distinctcellular responses. While TNF-R1 appears to be responsible for signalingmost TNF responses, the thymocyte proliferation stimulating activity ofTNF is specifically mediated by TNF-R2. In addition, TNF-R2 activatesthe transcription factor NF-KB (Lenardo & Baltimore, Cell 58: 227-2291989!) and mediates the transcriptional induction of thegranulocyte-macrophage colony stimulating factor (GM-CSF) gene (Miyatakeet al, EMBO J. 4: 2561-2568 1985!; Stanley et al, EMBO J. 4: 2569-25731985!) and the A20 zinc finger protein gene (Opipari et al, J. Biol.Chem. 265: 14705-14708 1990!) in CT6 cells. TNF-R2 also participates asan accessory component to TNF-R1 in the signaling of responses primarilymediated by TNF-R1, like cytotoxicity ( Tartaglia, L. A. and Goeddel, D.V., Immunol. Today 13, 151-153 1992!).

SUMMARY OF THE INVENTION

Although TNF itself, the TNF receptors and TNF activities mediated bythe two receptors have been studied extensively, the post-receptorsignal transduction mechanisms are unknown (see the review article byBeyaert, R. & Fiers, W., "Molecular mechanisms of tumor necrosisfactor-induced cytotoxicity: what we do understand and what we do not",FEBS Letters 340, 9-16 (1994)). This is especially true for the veryfirst step in the TNF receptor signal transduction cascade, i.e. for thequestion of how the membrane-bound receptor sends a signal into the cellafter activation by the ligand, TNF.

The present invention is based on the hypothesis that polypeptidefactors associated with the intracellular domain of TNF-R2 exist andparticipate in the TNF-R2 signal transduction cascade. Morespecifically, this invention is based on research directed to theidentification and isolation of native polypeptide factors that arecapable of association with the intracellular domain of TNF-R2 andparticipate in the intracellular post-receptor signaling of TNFbiological activities.

It is known that the TNF induced proliferation of murine CT6 cells ismediated by TNF-R2 (Tartaglia et al, 1991!, supra). To identify factorsthat are associated with the intracellular domain of hTNF-R2, thereceptor was immunoprecipitated from lysates of ³⁵ S!-labeledtransfected CT6 cells and from unlabeled transfected human embryonickidney 293 cells, which were then incubated with labeled lysate fromuntransfected CT6 cells. Several polypeptides with apparent molecularweights of 45-50 kD and one with an approximate molecular weight of 68kD were specifically coprecipitated with the immunoprecipitated hTNF-R2.These are hereinafter collectively referred to as tumor necrosis factorreceptor associated polypeptides, or TRAFs. Of the factors identifiedtwo have so far been purified and cloned. These two factors aredesignated as tumor necrosis factor receptor associated factors 1 and 2(TRAF1 and TRAF2; SEQ. ID. NOs: 2 and 4). A comparison of the amino acidsequences of TRAF1 and TRAF2 revealed that they share a high degree ofamino acid identity in their C-terminal domains (53% identity over 230amino acids), while their N-terminal domains are unrelated. These newfactors are believed to play a key role in the post-receptor signalingof TNF. Since the intracellular domain of TNF-R2 does not display anysequence homology to any other known receptor or protein, thesesignaling molecules might represent a novel signal transductionmechanism, the understanding of which can greatly contribute to thedevelopment of new strategies to improve the therapeutic value of TNF.

In one aspect, the present invention concerns a family of novel factors(TRAFs) capable of specific association with the intracellular domain ofa native TNF-R2. The invention specifically concerns tumor necrosisfactor receptor associated factors 1 and 2 (TRAF1 and TRAF2, SEQ. ID.NOs. 2 and 4), including the native factors from any human or non-humananimal species and their functional derivatives.

In another aspect, the invention concerns an isolated nucleic acidmolecule comprising a nucleotide sequence encoding a TRAF polypeptide.

In yet another aspect, the invention concerns an expression vectorcomprising the foregoing nucleic acid molecule operably linked tocontrol sequences recognized by a host cell transformed with the vector.

In a further aspect, the invention concerns a host cell transformed withthe foregoing expression vector.

In a still further aspect, the invention concerns molecules (includingpolypeptides, e.g. antibodies and TRAF analogs and fragments, peptidesand small organic molecules) which disrupt the interaction of a TNF-R2receptor associated factor and TNF-R2.

The invention specifically concerns antibodies, capable of specificbinding to a native TRAF polypeptide, and hybridoma cell lines producingsuch antibodies.

In a different aspect, the invention concerns a method of using anucleic acid molecule encoding a TRAF polypeptide as hereinabovedefined, comprising expressing such nucleic acid molecule in a culturedhost cell transformed with a vector comprising said nucleic acidmolecule operably linked to control sequences recognized by the hostcell transformed with the vector, and recovering the encoded polypeptidefrom the host cell.

The invention further concerns a method for producing a TRAF polypeptideas hereinabove defined, comprising inserting into the DNA of a cellcontaining nucleic acid encoding said polypeptide a transcriptionmodulatory element in sufficient proximity and orientation to thenucleic acid molecule to influence the transcription thereof.

The invention also provides a method of determining the presence of aTRAF polypeptide, comprising hybridizing DNA encoding such polypeptideto a test sample nucleic acid and determining the presence of TRAFpolypeptide DNA.

In a further aspect, the invention concerns an isolated nucleic acidmolecule encoding a fusion of an intracellular domain sequence of anative TNF-R2 and the DNA-binding domain of a transcriptional activator.

In a still further aspect, the invention concerns an isolated nucleicacid molecule encoding a fusion of a TRAF to the activation domain of atranscriptional activator.

The invention further concerns hybrid (fusion) polypeptides encoded bythe foregoing nucleic acids.

The invention also covers vectors comprising one or both of the nucleicacid molecules encoding the foregoing fusion proteins.

In a different aspect, the invention concerns an assay for identifying afactor capable of specific binding to the intracellular domain of anative TNF-R2, comprising

(a) expressing, in a single host cell carrying a reporter gene, nucleicacid molecules encoding a polypeptide comprising a fusion of anintracellular domain sequence of a native TNF-R2 to the DNA-bindingdomain of a transcriptional activator, and a fusion of a candidatefactor to the activation domain of a transcriptional activator; and

(b) monitoring the binding of the candidate factor to the TNF-R2intracellular domain sequence by detecting the molecule encoded by thereporter gene.

The invention further relates to an assay for identifying a factorcapable of specific association with the intracellular domain of anative TNF-R2, comprising

(a) expressing nucleic acid molecules encoding a polypeptide comprisinga fusion of an intracellular domain sequence of a native TNF-R2 to theDNA-binding domain of a transcriptional activator, and a secondpolypeptide comprising a fusion of a candidate polypeptide factor to theactivation domain of a transcriptional activator, in a single host celltransfected with nucleic acid encoding a polypeptide factor capable ofspecific binding to said TNF-R2, and with nucleic acid encoding areporter gene; and

(b) monitoring the association of said candidate factor with said TNF-R2or with said polypeptide factor capable of specific binding to saidTNF-R2 by detecting the polypeptide encoded by said reporter gene.

In a further aspect, the invention concerns an assay for identifying amolecule capable of disrupting the association of a TRAF with theintracellular domain of a native TNF-R2, comprising contacting a cellexpressing 1. a fusion of an intracellular domain sequence of a nativeTNF-R2 to the DNA-binding domain of a transcriptional activator, 2. afusion of a native TRAF polypeptide to the activation domain of saidtranscriptional activator, and 3. a reporter gene, with a candidatemolecule, and monitoring the ability of said candidate molecule todisrupt the association of said TRAF and TNF-R2 intracellular domainsequence by detecting the molecule encoded by the reporter gene. Thecell, just in the previous assays is preferably a yeast cell.

In addition to the "two-hybrid" format described above, the assay may beperformed in any conventional binding/inhibitor assay format. Forexample, one binding partner (TNF-R2 or TRAF) may be immobilized, andcontacted with the other binding partner equipped with a detectablelabel, such as a radioactive label, e.g. ³² P and the binding(association) of the two partners is detected in the presence of acandidate inhibitor. The design of a specific binding assay is wellwithin the skill of a person skilled in the art.

In a different aspect, the invention concerns a method of amplifying anucleic acid test sample comprising priming a nucleic acid polymerasereaction with nucleic acid encoding a TRAF polypeptide, as definedabove.

In another aspect, the invention concerns a method for detecting anucleic acid sequence coding for a polypeptide molecule which comprisesall or part of a TRAF polypeptide or a related nucleic acid sequence,comprising contacting the nucleic acid sequence with a detectable markerwhich binds specifically to at least part of the nucleic acid sequence,and detecting the marker so bound.

In yet another aspect, the invention concerns a method for treating apathological condition associated with a TNF biological activitymediated, fully or partially, by TNF-R2, comprising administering to apatient in need a therapeutically effective amount of a TRAF or amolecule Capable of disrupting the interaction of a TRAF and TNF-R2.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Activation of the transcription factor NF-kB through TNF-R2 inCT6 cells.

6 μg of nuclear extract prepared from CT6 cells that had been stimulatedfor 20 min with a 1:500 dilution of anti-mTNF-R2 polyclonal antibodiesor the respective preimmune serum were incubated with a radiolabeleddouble-stranded oligonucleotide containing either two wild-type (wt) ormutant (mr) NF-kB binding sites and analyzed for the induction of NF-kBDNA-binding activity by electrophoretic mobility shift assay (Schutze etal., Cell 71, 765-776 1992!). Binding reactions were either performedwithout competitor oligonucleotides or in the presence of a 500 foldexcess of unlabeled competitor oligonucleotides containing mutant NF-kBbinding sites or a binding site for the transcription factor AP-1(Angel, P. et al., Mol. Cell. Biol. 7: 2256-2266 1987!). F and B referto free oligonucleotide probe and oligonucleotide probe in a complexwith protein, respectively.

FIG. 2. Immunoprecipitation of hTNF-R2.

(A) ³⁵ S-labeled CT6 cells or CT6 cells expressing the hTNF-R2 werestimulated for 10 rain with 100 ng/ml hTNF or left untreated. The cellswere lysed and the hTNF-R2 immunoprecipitated as described in the textand analyzed by SDS-PAGE and autoradiography. The asterisk marks theband corresponding to the 75-80 kd hTNF-R2.

(B) The hTNF-R2 was immunoprecipitated from unstimulated orTNF-stimulated 293 or 293/TNF-R2 cells and incubated with lysates from³⁵ S-labeled CT6 cells. Arrows indicate bands of 45-50 kd and 68 kd thatcoprecipitate specifically with the hTNF-R2 in both experiments.Molecular weight markers are indicated on the right in kd.

FIG. 3. Purification of GST-hTNF-R2icd fusion protein.

Glutathione-S-transferase (GST) and GST-hTNF-R2icd fusion protein wereexpressed in E. coli, purified as described in the text and analyzed bySDS-PAGE and Coomassie staining. Molecular weight markers are indicatedon the right in kd.

FIG. 4. Coprecipitation of GST-hTNF-R2icd fusion protein in CT6 cellextracts. GST and GST-hTNF-R2icd fusion protein beads were incubatedwith lysates from ³⁵ S-labeled CT6 cells as described in the text andanalyzed by SDS-PAGE and autoradiography. Arrows indicate bands of 45-50kd and 68 kd that coprecipitate specifically with the GST-hTNF-R2icdfusion protein. Molecular weight markers are indicated on the right inkd.

FIG. 5. Coprecipitation of GST-mutant hTNF-R2icd fusion proteins in CT6cell extracts.

GST and GST-fusion proteins containing mutant intracellular domains ofthe hTNF-R2 were coupled to glutathione-agarose beads, incubated withlysates from ³⁵ S-labeled CT6 cells and analyzed by SDS-PAGE andautoradiography. Arrows indicate bands of 45-50 kd and 68 kd thatcoprecipitate specifically with the GST-fusion proteins containing thewild type (wt), the mutant -16, the Δ304-345 and the 384-424intracellular domains of hTNF-R2 but are not associated with the mutant-37 and -59 intracellular domains. Note that the pattern of these bandsis compressed in some cases due to the unlabeled fusion proteinsmigrating at the same size. Molecular weight markers are indicated onthe right in kd.

FIGS. 6A and 6B. Competition of TNF-R2 associated factors withGST-hTNF-R2icd fusion proteins.

(A) The hTNF-R2 was immunoprecipitated from 293 and 293/TNF-R2 cells andincubated with lysates from ³⁵ S-labeled CT6 cells that had beenpreincubated with 50 μl of the indicated GST-hTNF-R2icd fusion proteinbeads as competitor. Reactions were analyzed by SDS-PAGE andautoradiography. Arrows indicate bands of 45-50 kd and 68 kd thatcoprecipitate specifically with the hTNF-R2 and that are depleted bypreincubation with GST-fusion proteins containing the wild type (wt) andthe mutant -16 intracellular domains of hTNF-R2 but not by preincubationwith the mutant -37 and -59 intracellular domain fusion proteins.

(B) The 68 kd region of a similar experiment as described in (A) isshown. Molecular weight markers are indicated on the right in kd.

FIG. 7. Coprecipitation of GST-hTNF-R2icd fusion protein in Jurkat cellextracts. GST and GST-hTNF-R2icd fusion protein beads were incubatedwith lysates from ³⁵ S-labeled Jurkat cells that had been stimulated for10 min with 100 ng/ml hTNF or left untreated. Reactions were analyzed bySDS-PAGE and autoradiography. Arrows indicate bands of 45-50 kd, 67 kdand 73-75 kd that coprecipitate specifically with the GST-hTNF-R2icdfusion protein. Molecular weight markers are indicated on the right inkd.

FIG. 8. Subcellular localization of TNF-R2 associated factors.

Cytoplasmic and cell membrane fractions were prepared from ³⁵ S-labeledCT6 cells as described in the text. These fractions and a detergent(total) extract form CT6 cells were incubated with GST andGST-hTNF-R2icd fusion beads, and the reactions analyzed by SDS-Page andautoradiography. Arrows indicate bands of 45-50 kd and 68 kd thatcoprecipitate specifically with the GST-hTNF-R2icd fusion protein.Molecular weight markers are indicated on the right in kd.

FIG. 9. Purification of TNF-R2 associated factors.

Large scale purification of TNF-R2 associated factors from CT6 cells byGST-hTNF-R2icd fusion protein affinity chromatography was performed asdescribed in the text. One tenth of the obtained material was analyzedby SDS-PAGE and silver staining. Arrows indicate bands of 45-50 kd and68-70 kd that were eluted specifically from the GST-hTNF-R2icd fusionprotein affinity column. Molecular weight markers are indicated on theright in kd.

FIGS. 10A and 10B. Nucleotide and predicted amino acid sequence of theTRAF1 cDNA (SEQ. ID. NOS: 1 and 2).

The nucleic acid sequence of the TRAF1 cDNA is shown with numberingstarting from the first base after the Sa/I cloning linker. The deducedprotein sequence is displayed above with numbering from the initiationmethionine. In-frame stop codons upstream of the initiation methionineare underlined. Amino acids identified by sequencing the purified TRAF1protein are indicated in bold. The TRAF domain (see text) comprisesamino acids 180 (>) -409 (<). The potential leucine zipper region (seetext) extends between amino acids 183 (+) -259 (-). Amino acids withinthis region defining the heptade motif are indicated in italic.

FIGS. 11A and 11B. Nucleotide and predicted amino acid sequence of theTRAF2 cDNA (SEQ. ID. NOS: 3 and 4).

The nucleic acid sequence of the longest TRAF2 cDNA is shown withnumbering starting from the first base after the Sa/I cloning linker. Inaddition, the first nucleotide of four independently isolated pPC86 cDNAinserts (*) and the longest λ phage cDNA insert ( ) is indicated. Thededuced protein sequence is displayed above with numbering from theputative initiation methionine, which is in-frame with the GAL4activation domain coding region in all isolated pPC86TRAF2 cDNA clones(see text). Cysteine and histidine residues defining the RING fingermotif and the two TFIIIA-like zinc finger motifs (see text) areindicated in bold or underlined, respectively. The TRAF domain (seetext) comprises amino acids 272 (>) -501 (<). The potential leucinezipper region (see text) extends between amino acids 275 (+) -351 (-).Amino acids within this region defining the heptade motif are indicatedin italic.

FIGS. 12A and 12B. Sequence similarity of regions in TRAF2 tozinc-binding motifs.

(A) Comparison of amino acid sequences containing RING finger motifs.The TRAF2 RING finger motif is aligned with the respective zinc-bindingmotifs of the regulatory protein COP1 from A. thaliana (Deng et al.,Cell 71, 791-801 1992!; SEQ. ID. NO: 5), the human estrogen-responsivefinger protein EFP (Inoue et al., Proc. Natl. Acad. Sci. USA 90,11117-11121 1993!; SEQ. ID. NO: 6), the RAD18 and UVS-2 gene productsrequired for DNA repair in S. cerevisiae and N. crassa, respectively(Jones et al., Nucl. Acids Res. 16, 7119-7131 1988!; SEQ. ID. NO: 7;Tomita et al., Mol. Gert. Genet. 238, 225-233 1993!; SEQ. ID. NO: 8),the human V(D)J recombination activating gene product RAG-1 (Schatz etal, Cell 59, 1035-1048 1989!; SEQ. ID. NO: 9), the human 52 kdriboculeoprotein SS-A/Ro (Chan et al, J. Clin. Invest. 87, 68-76 1987!;Itoh et al., J. Clin. Invest. 87, 177-186 1987!; A⁵² in ref. 1 is p⁵² inref. 2; SEQ. ID. NO: 10); human RING1 (Lovering, GBTRANS accessionnumber Z14000 1992!; SEQ. ID. NO: 11), mouse T lymphocyte regulatoryprotein RPT-1 (Patarca et al, Proc. Natl. Acad. Sci. USA 85, 2733-27371988!; SEQ. ID.NO: 12), human regulatory protein RFP (Takahashi et al,Mol. Cell. Biol. 8, 1853-1856 1988!; SEQ. ID. NO: 13), and the productof the human proto-oncogen c-cbl (Blake et al., Oncogene 6, 653-6571991!; SEQ. ID. NO: 14).

(B) Comparison of amino acid sequences containing TFIIIA-type zincfinger motifs. A region in TRAF2 comprising two contiguous repeats ofthe consensus sequence ^(C) /_(H) -X₂₋₄ -^(C) /_(H) -X₂₋₁₅ -^(C) /_(H)-X₂₋₄ -^(C) /_(H) (Berg, J. Biol. Chem. 265, 6513-6516 1990!) is alignedwith similar zinc-binding motifs of the developmentally regulated DG 17gene product from D. discoideum (Driscoll & Williams, Mol. Cell. Biol.7, 4482-4489 1987!; SEQ. ID. NO: 15), the transcription factor IIIA formX. laevis (Miller et al., EMBO J. 4, 1609-1614 1985!; SEQ. ID. NO: 16),the Xenopus zinc finger proteins XLCOF14 and XFIN (Nietfeld et al., J.Mol. Biol. 208, 639-659 1989!; SEQ. ID. NO: 17; Ruiz i Altaba et al,EMBO J. 6, 3065-3070 1987!; SEQ. ID. NO: 18), the mouse ZFY1/2 and MFG2gene products (Mardon & Page, Cell 56, 765-770 1989!; SEQ. ID. NO: 19;Passananti et al, Proc. Natl. Acad. Sci. USA 86, 9421-9471 1989!; SEQ.ID. NO: 20), and the RAD18 and UVS-2 proteins (see above; SEQ. ID. NOS:21 and 22).

FIG. 13. Homology between TRAF1 and TRAF2.

An optimized alignment of the protein sequences of TRAF1 and TRAF2 isshown. Identical amino acids are boxed. The C-terminal TRAF domain (seetext) comprises amino acids 180-409 of TRAF1 and 272-501 of TRAF2.

FIGS. 14A and 14B. Hydropathy analysis of TRAF1 and TRAF2.

Hydropathy profiles of the amino acid sequences of TRAF1 (A) and TRAF2(B) were obtained by the method of Kyte and Doolittle, J. Mol. Biol.157, 105-132 (1982) using a window of twenty amino acids. The numbersunder each plot indicate positions of the amino acids of the respectiveprotein.

FIGS. 15A and 15B. Northern blot analysis of TRAF1 and TRAF2 mRNA.

(A) Northern blot analysis of TRAF1 and TRAF2 mRNA in CT6 cells. Thereis 3 μg of poly(A)⁺ RNA from CT6 cells per lane.

(B) Northern blot analysis of TRAF1 and TRAF2 mRNA in mouse tissues.Mouse multiple tissue northern blots (Clontech) were hybridized withradiolabeled TRAF1 and TRAF2 probes as described in the text.

FIG. 16. Coprecipitation of GST-TRAF2 fusion protein in 293 cellextracts. GST and GST-TRAF2 fusion protein beads were incubated withlysates from 293 and 293/TNF-R2 cells as described in the text.Reactions were analyzed by SDS-PAGE and Western blot analysis usinganti-human TNF-R1 monoclonal antibody 986 (0.5 μg/ml) and anti-humanTNF-R2 monoclonal antibody 1036 (0.5 μg/ml). An arrow indicates the75-80 kd hTNF-R2 band that is coprecipitated specifically with theGST-TRAF2 fusion protein. Molecular weight markers are indicated on theright in kd.

DETAILED DESCRIPTION OF THE INVENTION A. Definitions

The phrases "factor," "tumor necrosis factor receptor associatedfactor", "TNF-R2 associated factor" and "TRAF" are used interchangeablyand refer to a native factor capable of specific association with theintracellular domain of a native TNF-R2, and functional derivatives ofsuch native factor. In the context of this definition the phrase"specific association" is used in the broadest sense, and includesdirect binding to a site or region within the intracellular domain of anative TNF-R2 of the human or of any animal species, and indirectassociation with a native TNF-R2 intracellular domain, mediated by afurther molecule, such as another TRAF. The phrase "native TRAF"designates a TRAF polypeptide as occurring in nature in any cell type ofany human or non-human animal species, with or without the initiatingmethionine, whether purified from native source, synthesized, producedby recombinant DNA technology or by any combination of these and/orother methods. Native TRAFs specifically include monomeric, homo- andheterodimeric and homo- and heterooligomeric forms of such naturallyoccurring polypeptides. The native TRAF polypeptides preferably share anovel sequence motif in the C-terminal portion of their amino acidsequences, and preferably are at least about 40%, more preferably atleast about 50%, most preferably at least about 55% homologous withinthis C-terminal "TRAF domain". The "TRAF domain" encompasses about aminoacids 272 to 501 of the native mouse TRAF2 amino acid sequence, aboutamino acids 180 to 409 of the native mouse TRAF1 amino acid sequence,and homologous domains of other native TRAFs and their functionalderivatives.

The terms "native type 2 TNF receptor" and "native TNF-R2" are usedinterchangeably, and refer to any naturally occurring (native) type 2TNF receptor from any (human and non-human) animal species, with orwithout the initiating methionine and with or without a signal sequenceattached to the N-terminus, whether purified from native source,synthesized, produced by recombinant DNA technology or by anycombination of these and/or other methods.

The terms "native human type 2 TNF receptor" and "native human TNF-R2",which are used interchangeably, refer to a human TNF-R2 having the aminoacid sequence disclosed in EP 418,014 (published 20 Mar. 1991), with orwithout the initiating methionine and with or without a signal sequenceattached to the N-terminus, whether purified from native source,synthesized, produced by recombinant DNA technology or by anycombination of these and/or other methods, and other naturally occurringhuman TNF-R2 variants, including soluble and variously glycosylatedforms of native full-length human TNF-R2, whether purified from naturalsources, synthetically produced in vitro or obtained by geneticmanipulation including methods of recombinant DNA technology.

A "functional derivative" of a native polypeptide is a compound having aqualitative biological activity in common with the native polypeptide.Thus, a functional derivative of a native TRAF polypeptide is a compoundthat has a qualitative biological activity in common with a native TRAF."Functional derivatives" include, but are not limited to, fragments ofnative polypeptides from any animal species (including humans), andderivatives of native (human and non-human) polypeptides and theirfragments, provided that they have a biological activity in common witha respective native polypeptide. "Fragments" comprise regions within thesequence of a mature native polypeptide. The term "derivative" is usedto define amino acid sequence and glycosylation variants, and covalentmodifications of a native polypeptide, whereas the term "variant" refersto amino acid sequence and glycosylation variants within thisdefinition. Preferably, the functional derivatives are polypeptideswhich have at least about 65% amino acid sequence identity, morepreferably about 75% amino acid sequence identity, even more preferablyat least about 85% amino acid sequence identity, most preferably atleast about 95% amino acid sequence identity with the sequence of acorresponding native polypeptide. Most preferably, the functionalderivatives of a native TRAF polypeptide retain or mimic the region orregions within the native polypeptide sequence that directly participatein the association with the TNF-R2 intracellular domain and/or in homo-or heterodimerization. The phrase "functional derivative" specificallyincludes peptides and small organic molecules having a qualitativebiological activity in common with a native TRAF.

The term "biological activity" in the context of the definition offunctional derivatives is defined as the possession of at least oneadhesive, regulatory or effector function qualitatively in common with anative polypeptide (e.g. TRAF). A preferred biological property of thefunctional derivatives of the native TRAF polypeptides herein is theirability to associate with the intracellular domain of a native TNF-R2(either by direct binding or via interaction with another TRAF), andthereby mediate or block a biological response signaled (exclusively orpartially) by the TNF-R2 with which they are associated.

"Identity" or "homology" with respect to a native polypeptide and itsfunctional derivative is defined herein as the percentage of amino acidresidues in the candidate sequence that are identical with the residuesof a corresponding native polypeptide, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent homology,and not considering any conservative substitutions as part of thesequence identity. Neither N- or C-terminal extensions nor insertionsshall be construed as reducing identity or homology. Methods andcomputer programs for the alignment are well known in the art.

The TRAF polypeptides of the present invention specifically includenative murine TRAF-1 (SEQ. ID. NO: 2) and native murine TRAF-2 (SEQ. ID.NO: 4) their homo- and heterodimeric and homo- and heterooligomericforms, and their analogs in other mammalian species, such as rat,porcine, equine, cow, higher primates, and humans, and the functionalderivatives of such native polypeptides. The functional derivatives of anative TRAF-1 or native TRAF-2 receptor are preferably encoded by DNAscapable, under stringent conditions, of hybridizing to the complement ofa DNA encoding a native TRAF polypeptide. More preferably, thefunctional derivatives share at least about 40% sequence homology, morepreferably at least about 50% sequence homology, even more preferably atleast about 55% sequence homology, most preferably at least about 60%sequence homology with any domain, and preferably with the TNF-R2binding domain(s) and/or the dimerization domain(s), of a native TRAFpolypeptide. In a most preferred embodiment, a functional derivativewill share at least about 50% sequence homology with the C-terminal TRAFregion of murine TRAF2, or are encoded by DNA capable of hybridizing,under stringent conditions, with the complement of DNA encoding the TRAFregion of murine TRAF2.

The "stringent conditions" are overnight incubation at 42° C. in asolution comprising: 20% formamide, 5× SSC (150 mM NaCl, 15 mM trisodiumcitrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10%dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA.

The terms "amino acid" and "amino acids" refer to all naturallyoccurring L-α-amino acids. The amino acids are identified by either thesingle-letter or three-letter designations:

    ______________________________________                                        Asp  D       aspartic acid                                                                             IIe  I     isoleucine                                Thr  T       threonine   Leu  L     leucine                                   Ser  S       serine      Tyr  Y     tyrosine                                  Glu  E       glutamic acid                                                                             Phe  F     phenylalanine                             Pro  P       proline     His  H     histidine                                 Gly  G       glycine     Lys  K     lysine                                    Ala  A       alanine     Arg  R     arginine                                  Cys  C       cysteine    Trp  W     tryptophan                                Val  V       valine      Gln  Q     glutamine                                 Met  M       methionine  Asn  N     asparagine                                ______________________________________                                    

These amino acids may be classified according to the chemicalcomposition and properties of their side chains. They are broadlyclassified into two groups, charged and uncharged. Each of these groupsis divided into subgroups to classify the amino acids more accurately:

I. Charged Amino Acids

Acidic Residues: aspartic acid, glutamic acid

Basic Residues: lysine, arginine, histidine

II. Uncharged Amino Acids

Hydrophilic Residues: serine, threonine, asparagine, glutamine

Aliphatic Residues: glycine, alanine, valine, leucine, isoleucine

Non-polar Residues: cysteine, methionine, proline

Aromatic Residues: phenylalanine, tyrosine, tryptophan

The term "amino acid sequence variant" refers to molecules with somedifferences in their amino acid sequences as compared to a native aminoacid sequence.

Substitutional variants are those that have at least one amino acidresidue in a native sequence removed and a different amino acid insertedin its place at the same position. The substitutions may be single,where only one amino acid in the molecule has been substituted, or theymay be multiple, where two or more amino acids have been substituted inthe same molecule.

Insertional variants are those with one or more amino acids insertedimmediately adjacent to an amino acid at a particular position in anative sequence. Immediately adjacent to an amino acid means connectedto either the α-carboxy or α-amino functional group of the amino acid.

Deletional variants are those with one or more amino acids in the nativeamino acid sequence removed. Ordinarily, deletional variants will haveone or two amino acids deleted in a particular region of the molecule.

The term "glycosylation variant" is used to refer to a glycoproteinhaving a glycosylation profile different from that of a nativecounterpart or to glycosylated variants of a polypeptide unglycosylatedin its native form(s). Glycosylation of polypeptides is typically eitherN-linked or O-linked. N-linked refers to the attachment of thecarbohydrate moiety to the side-chain of an asparagine residue. Thetripeptide sequences, asparagine-X-serine and asparagine-X-threonine,wherein X is any amino acid except proline, are recognition sequencesfor enzymatic attachment of the carbohydrate moiety to the asparagineside chain. O-linked glycosylation refers to the attachment of one ofthe sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyaminoacid, most commonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be involved in O-linked glycosylation.

Antibodies (Abs) and immunoglobulins (Igs) are glycoproteins having thesame structural characteristics. While antibodies exhibit bindingspecificity to a specific antigen, immunoglobulins include bothantibodies and other antibody-like molecules which lack antigenspecificity. Polypeptides of the latter kind are, for example, producedat low levels by the lymph system and at increased levels by myelomas.

Native antibodies and immunoglobulins are usually heterotetramericglycoproteins of about 150,000 daltons, composed of two identical light(L) chains and two identical heavy (H) chains. Each light chain islinked to a heavy chain by one covalent disulfide bond, while the numberof disulfide linkages varies between the heavy chains of differentimmunoglobulin isotypes. Each heavy and light chain also has regularlyspaced intrachain disulfide bridges. Each heavy chain has at one end avariable domain (V_(H)) followed by a number of constant domains. Eachlight chain has a variable domain at one and (V_(L)) and a constantdomain at its other end; the constant domain of the light chain isaligned with the first constant domain of the heavy chain, and the lightchain variable domain is aligned with the variable domain of the heavychain. Particular amino acid residues are believed to form an interfacebetween the light and heavy chain variable domains (Clothia et al., J.Mol. Biol. 186, 651-663 1985!; Novotny and Haber, Proc. Natl. Acad. Sci.USA 82, 4592-4596 1985!).

The term "variable" refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthrough the variable domains of antibodies. It is concentrated in threesegments called complementarity determining regions (CDRs) orhypervariable regions both in the light chain and the heavy chainvariable domains. The more highly conserved portions of variable domainsare called the framework (FR). The variable domains of native heavy andlight chains each comprise four FR regions, largely adopting a β-sheetconfiguration, connected by three CDRs, which form loops connecting, andin some cases forming part of, the β-sheet structure. The CDRs in eachchain are held together in close proximity by the FR regions and, withthe CDRs from the other chain, contribute to the formation of theantigen binding site of antibodies (see Kabat, E. A. et al., Sequencesof Proteins of Immunological Interest, National Institute of Health,Bethesda, Md. 1991!). The constant domains are not involved directly inbinding an antibody to an antigen, but exhibit various effectorfunctions, such as participation of the antibody in antibody-dependentcellular toxicity.

Papain digestion of antibodies produces two identical antigen bindingfragments, called Fab fragments, each with a single antigen bindingsite, and a residual "Fc" fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab')₂ fragment thathas two antigen combining sites and is still capable of cross-linkingantigen.

"Fv" is the minimum antibody fragment which contains a complete antigenrecognition and binding site. This region consists of a dimer of oneheavy and one light chain variable domain in tight, non-covalentassociation. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen binding site on thesurface of the V_(H) -V_(L) dimer. Collectively, the six CDRs conferantigen binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab' fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab'-SH is the designationherein for Fab' in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab')₂ antibody fragments originally wereproduced as pairs of Fab' fragments which have hinge cysteines betweenthem. Other, chemical couplings of antibody fragments are also known.

The light chains of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa and lambda (A), based on the amino acid sequences of theirconstant domains.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG andIgM, and several of these may be further divided into subclasses(isotypes), e.g. IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2. Theheavy chain constant domains that correspond to the different classes ofimmunoglobulins are called α, delta, epsilon, γ, and μ, respectively.The subunit structures and three-dimensional configurations of differentclasses of immunoglobulins are well known.

The term "antibody" is used in the broadest sense and specificallycovers single monoclonal antibodies (including agonist and antagonistantibodies), antibody compositions with polyepitopic specificity, aswell as antibody fragments (e.g., Fab, F(ab')₂, and Fv), so long as theyexhibit the desired biological activity.

The term "monoclonal antibody" as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. In addition to their specificity, the monoclonal antibodies areadvantageous in that they are synthesized by the hybridoma culture,uncontaminated by other immunoglobulins. The modifier "monoclonal"indicates the character of the antibody as being obtained from asubstantially homogeneous population of antibodies, and is not to beconstrued as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by the hybridoma method firstdescribed by Kohler & Milstein, Nature 256:495 (1975), or may be made byrecombinant DNA methods see, e.g. U.S. Pat. No. 4,816,567 (Cabilly etal.)!.

The monoclonal antibodies herein specifically include "chimeric"antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567(Cablily et al; Morrison et al., Proc. Natl. Acad. Sci. USA 81,6851-6855 1984!).

"Humanized" forms of non-human (e.g. murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab'. F(ab')₂ or other antigen-binding subsequences of antibodies)which contain minimal sequence derived from non-human immunoglobulin.For the most part, humanized antibodies are human immunoglobulins(recipient antibody) in which residues from a complementary determiningregion (CDR) of the recipient are replaced by residues from a CDR of anon-human species (donor antibody) such as mouse, rat or rabbit havingthe desired specificity, affinity and capacity. In some instances, Fvframework residues of the human immunoglobulin are replaced bycorresponding non-human residues. Furthermore, humanized antibody maycomprise residues which are found neither in the recipient antibody norin the imported CDR or framework sequences. These modifications are madeto further refine and optimize antibody performance. In general, thehumanized antibody will comprise substantially all of at least one, andtypically two, variable domains, in which all or substantially all ofthe CDR regions correspond to those of a non-human immunoglobulin andall or substantially all of the FR regions are those of a humanimmunoglobulin consensus sequence. The humanized antibody optimally alsowill comprise at least a portion of an immunoglobulin constant region(Fc), typically that of a human immunoglobulin. For further details see:Jones et al, Nature 321, 522-525 1986!; Reichmann et al., Nature 332,323-329 1988!; and Presta, Curr. Op. Struct. Biol. 2 593-596 1992!).

In the context of the present invention the expressions "cell", "cellline", and "cell culture" are used interchangeably, and all suchdesignations include progeny. It is also understood that all progeny maynot be precisely identical in DNA content, due to deliberate orinadvertent mutations. Mutant progeny that have the same function orbiological property, as screened for in the originally transformed cell,are included.

"Transformation" means introducing DNA into an organism so that the DNAis replicable, either as an extrachromosomal element or by chromosomalintegration.

"Transfection" refers to the taking up of an expression vector by a hostcell whether or not any coding sequences are in fact expressed.

The terms "transformed host cell" and "transformed" refer to theintroduction of DNA into a cell. The cell is termed a "host cell", andit may be a prokaryotic or a eukaryotic cell. Typical prokaryotic hostcells include various strains of E. coli. Typical eukaryotic host cellsare mammalian, such as Chinese hamster ovary cells or human embryonickidney 293 cells. The introduced DNA is usually in the form of a vectorcontaining an inserted piece of DNA. The introduced DNA sequence may befrom the same species as the host cell or a different species from thehost cell, or it may be a hybrid DNA sequence, containing some foreignand some homologous DNA.

The terms "replicable expression vector" and "expression vector" referto a piece of DNA, usually double-stranded, which may have inserted intoit a piece of foreign DNA. Foreign DNA is defined as heterologous DNA,which is DNA not naturally found in the host cell. The vector is used totransport the foreign or heterologous DNA into a suitable host cell.Once in the host cell, the vector can replicate independently of thehost chromosomal DNA, and several copies of the vector and its inserted(foreign) DNA may be generated. In addition, the vector contains thenecessary elements that permit translating the foreign DNA into apolypeptide. Many molecules of the polypeptide encoded by the foreignDNA can thus be rapidly synthesized.

"Oligonucleotides" are short-length, single- or double-strandedpolydeoxynucleotides that are chemically synthesized by known methodssuch as phosphotriester, phosphite, or phosphoramidite chemistry, usingsolid phase techniques such as those described in EP 266,032, published4 May 1988, or via deoxynucleoside H-phosphanate intermediates asdescribed by Froehler et al., Nucl. Acids Res. 14, 5399 (1986)!. Theyare then purified on polyacrylamide gels.

B. Identification and Purification of TRAFs

The native TRAF polypeptides can be identified in and purified fromcertain tissues known to possess a type 2 TNF receptor (TNF-R2) mRNA andto express it at a detectable level. Thus, murine TRAF can, for example,be obtained from the murine interleukin 2 (IL-2)-dependent cytotoxic Tcell line CT6 (Ranges et al J. Immunol. 142, 1203-1208 1989!). MurineTRAF1 can also be purified from spleen, lung and testis; whereas murineTRAF2 can be isolated and purified from an even larger variety oftissues, including heart, brain, spleen, lung, liver, skelet al muscle,kidney and testis (see FIG. 15b). In general, TRAF proteins are expectedto be expressed in human tissues that are known to express TNF-R2,although not all of such tissues will express all TRAFs. Alternatively,TRAF polypeptides can be isolated from cell lines transfected with DNAencoding a native TNF-R2 or a TNF-R2 derivative comprising intracellulardomain sequences participating in the interaction with TRAFs. Factorsthat are associated with the intracellular domain of a native TNF-R2 canbe identified by immunoprecipitation of the receptor or receptorderivative from cells expressing it. Immunoprecipitation in generalconsists of multiple ordered steps, including lysing the cell withdetergent if the TNF-R2 is membrane-bound, binding of TNF-R2 to ananti-TNF-R2 antibody, precipitating the antibody complex, washing theprecipitate, and dissociating TNF-R2 and any associated factor from theimmune complex. The dissociated factor(s) can then be analyzed byelectrophoretic methods. In a preferred embodiment, radiolabeled TNF-R2(or a derivative) is immunoprecipitated with protein A-agarose (OncogeneScience) or with protein A-Sepharose (Pharmacia). In this case, theTNF-R2/anti-TNF-R2 antibody immune complexes are precipitated byStaphylococcus aureus protein A bound to the agarose or Sepharose. Theimmunoprecipitate is then analyzed by autoradiography or byfiuorography, depending on the actual radiolabel used. The TRAF proteins(which are characterized by their ability to associate with theintracellular domain of TNF-R2) will coprecipitate with the receptor orreceptor derivative, and can be further purified by methods known in theart, such as purification on an affinity column.

A large-scale purification scheme for purifying factors that associatewith the intracellular domain of TNF-R2 takes advantage of plasmidexpression vectors that direct the synthesis of foreign polypeptides inE. coli as fusions with the C terminus of glutathione S-transferase(GST), as described by Smith, D. B. and Johnson, K. S., Gene 67 31-40(1988). The intracellular domain of TNF-R2 is expressed as a fusionprotein with GST in E. coli recombinant host cells, and can be purifiedfrom crude bacterial lysates by absorption on glutathione-agarose beads(Sigma). A cell lysate containing the factor(s) to be purified is thenapplied to a GST-TNF-R2 fusion protein affinity column. Protein(s) boundto the column is/are eluted, precipitated and isolated by SDS-PAGE underreducing conditions, and visualized by silver staining. GST gene fusionvectors (pGEX vectors) as well as kits for cloning and expression of GSTfusion systems are commercially available from Pharmacia (see PharmaciaCatalog, 1994, pages 133; and 142-143).

Purified protein can be either sequenced directly by automated Edmandegradation with a model 470A Applied Biosystems gas phase sequencerequipped with a 120A PTH amino acid analyzer or sequenced afterdigestion with various chemicals or enzymes. PTH amino acids wereintegrated using a ChromPerfect data system (Justice Innovations, PaloAlto, Calif.). Sequence interpretation can be performed on a VAX 11/785Digital Equipment Corporation computer as described by Henzel et al, J.Chromatoqraphy 404, 41 (1987). In some cases, eluates electrophoresed onSDS polyacrylamide gels are electrotransferred to a PVDF membrane(ProBlott, ABI, Foster City, Calif.) and stained with CoomassieBrilliant Blue R250 (Sigma). The specific protein is excised from theblot for N-terminal sequencing. To determine internal protein sequences,purified fractions obtained by reverse phase o capillary HPLC aretypically dried under vacuum (SpeedVac), resuspended in appropriatebuffers, and digested with cyanogen bromide, and/or various proteases,such as trypsin, the lysine-specific enzyme Lys-C (Wako Chemicals,Richmond, Va.) or Asp-N (Boehringer Mannheim, Indianapolis, Ind.). Afterdigestion, the resultant peptides are sequenced as a mixture or areresolved by HPLC.

C. Recombinant Production of TRAF Polypeptides

Preferably, the TRAF polypeptides are prepared by standard recombinantprocedures by culturing cells transfected to express TRAF polypeptidenucleic acid (typically by transforming the cells with an expressionvector) and recovering the polypeptide from the cells. However, it isenvisioned that the TRAF polypeptides may be produced by homologousrecombination, or by recombinant production methods utilizing controlelements introduced into cells already containing DNA encoding an TRAFpolypeptide. For example, a powerful promoter/enhancer element, asuppressor, or an exogenous transcription modulatory element may beinserted in the genome of the intended host cell in proximity andorientation sufficient to influence the transcription of DNA encodingthe desired TRAF polypeptide. The control element does not encode theTRAF polypeptide, rather the DNA is indigenous to the host cell genome.One next screens for cells making the polypeptide of this invention, orfor increased or decreased levels of expression, as desired.

Thus, the invention contemplates a method for producing a TRAFpolypeptide comprising inserting into the genome of a cell containingnucleic acid encoding a TRAF polypeptide a transcription modulatoryelement in sufficient proximity and brientation to the nucleic acidmolecule to influence transcription thereof, with an optional furtherstep of culturing the cell containing the transcription modulatoryelement and the nucleic acid molecule. The invention also contemplates ahost cell containing the indigenous TRAF polypeptide nucleic acidmolecule operably linked to exogenous control sequences recognized bythe host cell.

1. Isolation of DNA encoding the TRAF polypeptides

For the purpose of the present invention, DNA encoding a TRAFpolypeptide can be obtained from cDNA libraries prepared from tissuebelieved to possess a type 2 TNF receptor (TNF-R2) mRNA and to expressit at a detectable level. For example, cDNA library can be constructedby obtaining polyadenylated mRNA from a cell line known to expressTNF-R2, and using the mRNA as a template to synthesize double strandedcDNA. Human and non-human cell lines suitable for this purpose have beenlisted hereinabove. It is noted, however, that TNF-R2 is known to beexpressed in a large variety of further tissues which can allpotentially serve as a source of TRAF cDNA, even though not all membersof the TRAF family will be expressed in all TNF-R2 expressing tissues.Alternatively, DNA encoding new TRAF polypeptides can be obtained fromcDNA libraries prepared from tissue known to express a previouslyidentified TRAF polypeptide at a detectable level. The TRAF polypeptidegenes can also be obtained from a genomic library, such as a humangenomic cosmid library.

Libraries, either cDNA or genomic, are screened with probes designed toidentify the gene of interest or the protein encoded by it. For cDNAexpression libraries, suitable probes include monoclonal and polyclonalantibodies that recognize and specifically bind to a TRAF polypeptide.For cDNA libraries, suitable probes include carefully selectedoligonucleotide probes (usually of about 20-80 bases in length) thatencode known or suspected portions of a TRAF polypeptide from the sameor different species, and/or complementary or homologous cDNAs orfragments thereof that encode the same or a similar gene. Appropriateprobes for screening genomic DNA libraries include, without limitation,oligonucleotides, cDNAs, or fragments thereof that encode the same or asimilar gene, and/or homologous genomic DNAs or fragments thereof.Screening the cDNA or genomic library with the selected probe may beconducted using standard procedures as described in Chapters 10-12 ofSambrook et al., Molecular Cloning: A Laboratory Manual, New York, ColdSpring Harbor Laboratory Press, 1989).

A preferred method of practicing this invention is to use carefullyselected oligonucleotide sequences to screen cDNA libraries from varioustissues. The oligonucleotide sequences selected as probes should besufficient in length and sufficiently unambiguous that false positivesare minimized. The actual nucleotide sequence(s) is/are usually designedbased on regions of a TRAF which have the least codon redundance. Theoligonucleotides may be degenerate at one or more positions. The use ofdegenerate oligonucleotides is of particular importance where a libraryis screened from a species in which preferential codon usage is notknown.

The oligonucleotide must be labeled such that it can be detected uponhybridization to DNA in the library being screened. The preferred methodof labeling is to use ATP (e.g., y³² P) and polynucleotide kinase toradiolabel the 5' end of the oligonucleotide. However, other methods maybe used to label the oligonucleotide, including, but not limited to,biotinylation or enzyme labeling.

cDNAs encoding TRAFs can also be identified and isolated by other knowntechniques of recombinant DNA technology, such as by direct expressioncloning or by using the polymerase chain reaction (PCR) as described inU.S. Pat. No. 4,683,195, issued 28 Jul. 1987, in section 14 of Sambrooket al, Molecular Cloning: A Laboratory Manual, second edition, ColdSpring Harbor Laboratory Press. New York, 1989, or in Chapter 15 ofCurrent Protocols in Molecular Biology, Ausubel et al eds., GreenePublishing Associates and Wiley-lnterscience 1991. This method requiresthe use of oligonucleotide probes that will hybridize to DNA encoding aTRAF.

According to a preferred method for practicing the invention, the codingsequences for TRAF proteins can be identified in a recombinant cDNAlibrary or a genomic DNA library based upon their ability to interactwith the intracellular domain of a TNF-R2. For this purpose one can usethe yeast genetic system described by Fields and co-workers (Fields andSong, Nature (London) 340, 245-246 1989!; Chien et al, Proc. Natl. Acad.Sci. USA 88, 9578-9582 1991!) as disclosed by Chevray and Nathans (Proc.Natl. Acad. Sci. USA 89, 5789-5793 1991!). Many transcriptionalactivators, such as yeast GAL4, consist of two physically discretemodular domains, one acting as the DNA-binding domain, while the otherone functioning as the transcription activation domain. The yeastexpression system described in the foregoing publications (generallyreferred to as the "two-hybrid system") takes advantage of thisproperty, and employs two hybrid proteins, one in which the targetprotein is fused to the DNA-binding domain of GAL4, and another, inwhich candidate activating proteins are fused to the activation domain.The expression of a GALl-lacZ reporter gene under control of aGAL4-activated promoter depends on reconstitution of GAL4 activity viaprotein-protein interaction. Colonies containing interactingpolypeptides are detected with a chromogenic substrate forβ-galactosidase. A complete kit (MATCHMAKER™) for identifyingprotein-protein interactions between two specific proteins using thetwo-hybrid technique is commercially available from Clontech. Thissystem can also be extended to map protein domains involved in specificprotein interactions as well as to pinpoint amino acid residues that arecrucial for these interactions.

To directly isolate genes encoding proteins that associate with theintracellular domain of TNF-R2, DNA encoding a TNF-R2 intracellulardomain or a fragment thereof is cloned into a vector containing DNAencoding the DNA-binding domain of GAL4. A plasmid cDNA library is thenconstructed by cloning double-stranded cDNA encoding a candidate factorin a vector comprising DNA encoding the GAL4 transcriptional activationdomain. Thereafter, yeast cells containing reporter genes arecotransformed with the TNF-R2-GAL4 DNA binding domain vector and withlibrary plasmid DNA. Typically, an S. cerevisiae cell containing tworeporter genes: lacZ(βgal) and His genes, serves as a host forcotransformation. Yeast transformants are selected by plating onsupplemented synthetic dextrose medium lacking tryptophan, leucine andhistidine, and protein-protein interactions are monitored by the yeastcolony filter β-galactosidase assay, essentially as described by Chevrayand Nathans, supra. Only colonies with protein-protein interaction willgrow on his' plates, and are then analyzed for β-gal as a furthercontrol.

Once cDNA encoding a TRAF from one species has been isolated, cDNAs fromother species can also be obtained by cross-species hybridization.According to this approach, human or other mammalian cDNA or genomiclibraries are probed by labeled oligonucleotide sequences selected fromknown TRAF sequences (such as murine TRAF-1 and TRAF-2 as disclosed inthe present application) in accord with known criteria, among which isthat the sequence should be sufficient in length and sufficientlyunambiguous that false positives are minimized. Typically, a ³²P-labeled oligonucleotide having about 30 to 50 bases is sufficient,particularly if the oligonucleotide contains one or more codohs formethionine or tryptophan. Isolated nucleic acid will be DNA that isidentified and separated from contaminant nucleic acid encoding otherpolypeptides from the source of nucleic acid.

Once the sequence is known, the gene encoding a particular TRAFpolypeptide can also be obtained by chemical synthesis, following one ofthe methods described in Engels and Uhlmann, Agnew. Chem. Int. Ed. Engl.28, 716 (1989). These methods include triester, phosphite,phosphoramidite and H-phosphonate methods, PCR and other autoprimermethods, and oligonucleotide syntheses on solid supports.

2. Amino Acid Sequence Variants of a native TRAF proteins or fragments

Amino acid sequence variants of native TRAFs and TRAF fragments areprepared by methods known in the art by introducing appropriatenucleotide changes into a native or variant TRAF DNA, or by in vitrosynthesis of the desired polypeptide. There are two principal variablesin the construction of amino acid sequence variants: the location of themutation site and the nature of the mutation. With the exception ofnaturally-occurring alleles, which do not require the manipulation ofthe DNA sequence encoding the TRAF, the amino acid sequence variants ofTRAF are preferably constructed by mutating the DNA, either to arrive atan allele or an amino acid sequence variant that does not occur innature.

One group of the mutations will be created within the domain or domainsidentified as being involved in the interaction with the intracellulardomain of TNF-R2. TRAF variants mutated to enhance their association(binding or indirect association) with TNF-R2 will be useful asinhibitors of native TNF-R2/native TNF interaction. In addition, suchvariants will be useful in the diagnosis of pathological conditionsassociation with the overexpression of TNF-R2, and in the purificationof TNF-R2. A target for such mutations is the N-terminal RING fingerdomain of TRAF2 and related factors, as this domain is believed to beinvolved in the interaction with the intracellular domain of TNF-R2.

Another group of mutations will be performed within region(s) involvedin interactions with other TNF-R2 associated factors. Thus, amino acidalterations within the homologous C-terminal domains (proteindimerization motif) of TRAF1, TRAF2 and other factors of the TRAF familycan enhance the ability of such factors to form stable dimers which arerequired for signaling through the TNF-R2 receptor.

Alternatively or in addition, amino acid alterations can be made atsites that differ in TRAF proteins from various species, or in highlyconserved regions, depending on the goal to be achieved.

Sites at such locations will typically be modified in series, e.g. by(1) substituting first with conservative choices and then with moreradical selections depending upon the results achieved, (2) deleting thetarget residue or residues, or (3) inserting residues of the same ordifferent class adjacent to the located site, or combinations of options1-3.

One helpful technique is called "alanine scanning" (Cunningham andWells, Science 244, 1081-1085 1989!). Here, a residue or group of targetresidues is identified and substituted by alanine or polyalanine. Thosedomains demonstrating functional sensitivity to the alaninesubstitutions are then refined by introducing further or othersubstituents at or for the sites of alanine substitution.

After identifying the desired mutation(s), the gene encoding a TRAFvariant can, for example, be obtained by chemical synthesis ashereinabove described.

More preferably, DNA encoding a TRAF amino acid sequence variant isprepared by site-directed mutagenesis of DNA that encodes an earlierprepared variant or a nonvariant version of the TRAF. Site-directed(site-specific) mutagenesis allows the production of TRAF variantsthrough the use of specific oligonucleotide sequences that encode theDNA sequence of the desired mutation, as well as a sufficient number ofadjacent nucleotides, to provide a primer sequence of sufficient sizeand sequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Typically, a primer of about 20 to 25nucleotides in length is preferred, with about 5 to 10 residues on bothsides of the junction of the sequence being altered. In general, thetechniques of site-specific mutagenesis are well known in the art, asexemplified by publications such as, Edelman et al., DNA 2, 183 (1983).As will be appreciated, the site-specific mutagenesis techniquetypically employs a phage vector that exists in both a single-strandedand double-stranded form. Typical vectors useful in site-directedmutagenesis include vectors such as the M13 phage, for example, asdisclosed by Messing et al, Third Cleveland Symposium on Macromoleculesand Recombinant DNA, A. Walton, ed., Elsevier, Amsterdam (1981). Thisand other phage vectors are commercially available and their use is wellknown to those skilled in the art. A versatile and efficient procedurefor the construction of oligodeoxyribonucleotide directed site-specificmutations in DNA fragments using M13-derived vectors was published byZoller, M. J. and Smith, M., Nucleic Acids Res. 10, 6487-6500 1982!).Also, plasmid vectors that contain a single-stranded phage origin ofreplication (Veira et al., Meth. Enzymol. 153, 3 1987!) may be employedto obtain single-stranded DNA. Alternatively, nucleotide substitutionsare introduced by synthesizing the appropriate DNA fragment in vitro,and amplifying it by PCR procedures known in the art.

In general, site-specific mutagenesis herewith is performed by firstobtaining a single-stranded vector that includes within its sequence aDNA sequence that encodes the relevant protein. An oligonucleotideprimer bearing the desired mutated sequence is prepared, generallysynthetically, for example, by the method of Crea et al., Proc. Natl.Acad. Sci. USA 75, 5765 (1978). This primer is then annealed with thesingle-stranded protein sequence-containing vector, and subjected toDNA-polymerizing enzymes such as, E. coli polymerase I Klenow fragment,to complete the synthesis of the mutation-bearing strand. Thus, aheteroduplex is formed wherein one strand encodes the originalnon-mutated sequence and the second strand bears the desired mutation.This heteroduplex vector is then used to transform appropriate hostcells such as JP101 cells, and clones are selected that includerecombinant vectors bearing the mutated sequence arrangement.Thereafter, the mutated region may be removed and placed in anappropriate expression vector for protein production.

The PCR technique may also be used in creating amino acid sequencevariants of a TRAF. When small amounts of template DNA are used asstarting material in a PCR, primers that differ slightly in sequencefrom the corresponding region in a template DNA can be used to generaterelatively large quantities of a specific DNA fragment that differs fromthe template sequence only at the positions where the primers differfrom the template. For introduction of a mutation into a plasmid DNA,one of the primers is designed to overlap the position of the mutationand to contain the mutation; the sequence of the other primer must beidentical to a stretch of sequence of the opposite strand of theplasmid, but this sequence can be located anywhere along the plasmidDNA. It is preferred, however, that the sequence of the second primer islocated within 200 nucleotides from that of the first, such that in theend the entire amplified region of DNA bounded by the primers can beeasily sequenced. PCR amplification using a primer pair like the onejust described results in a population of DNA fragments that differ atthe position of the mutation specified by the primer, and possibly atother positions, as template copying is somewhat error-prone.

If the ratio of template to product material is extremely low, the vastmajority of product DNA fragments incorporate the desired mutation(s).This product material is used to replace the corresponding region in theplasmid that served as PCR template using standard DNA technology.Mutations at separate positions can be introduced simultaneously byeither using a mutant second primer or performing a second PCR withdifferent mutant primers and ligating the two resulting PCR fragmentssimultaneously to the vector fragment in a three (or more) partligation.

In a specific example of PCR mutagenesis, template plasmid DNA (1 μg) islinearized by digestion with a restriction endonuclease that has aunique recognition site in the plasmid DNA outside of the region to beamplified. Of this material, 100 ng is added to a PCR mixture containingPCR buffer, which contains the four deoxynucleotide triphosphates and isincluded in the GeneAmp^(R) kits (obtained from Perkin-Elmer Cetus,Norwalk, Conn. and Emeryville, Calif.), and 25 pmole of eacholigonucleotide primer, to a final volume of 50 μl. The reaction mixtureis overlayered with 35 μl mineral oil. The reaction is denatured for 5minutes at 100° C., placed briefly on ice, and then 1 μl Thermusaquaticus (Taq) DNA polymerase (5 units/I), purchased from Perkin-ElmerCetus, Norwalk, Conn. and Emeryville, Calif.) is added below the mineraloil layer. The reaction mixture is then inserted into a DNA ThermalCycler (purchased from Perkin° Elmer Cetus) programmed as follows:

2 min. 55° C.,

30 sec. 72° C., then 19 cycles of the following:

30 sec. 94° C.,

30 sec. 55° C., and

30 sec. 72° C.

At the end of the program, the reaction vial is ranoved from the thermalcycler and the aqueous phase transferred to a new vial, extracted withphenol/chloroform (50:50 vol), and ethanol precipitated, and the DNA isrecovered by standard procedures. This material is subsequentlysubjected to appropriate treatments for insertion into a vector.

Another method for preparing variants, cassette mutagenesis, is based onthe technique described by Wells et al. Gene 34, 315 (1985)!. Thestarting material is the plasmid (or vector) comprising the TRAF DNA tobe mutated. The codon(s) within the TRAF to be mutated are identified.There must be a unique restriction endonuclease site on each side of theidentified mutation site(s). If no such restriction sites exist, theymay be generated using the above-described oligonucleotide-mediatedmutagenesis method to introduce them at appropriate locations in theTRAF DNA. After the restriction sites have been introduced into theplasmid, the plasmid is cut at these sites to linearize it. Adouble-stranded oligonucleotide encoding the sequence of the DNA betweenthe restriction site but containing the desired mutation(s) issynthesized using standard procedures. The two strands are synthesizedseparately and then hybridized together using standard techniques. Thisdouble-stranded oligonucleotide is referred to as the cassette. Thiscassette is designed to have 3' and 5' ends that are compatible with theends of the linearized plasmid, such that it can be directly ligated tothe plasmid. This plasmid now contains the mutated TRAF DNA sequence.

Additionally, the so-called phagemid display method may be useful inmaking amino acid sequence variants of native or variant TRAFs or theirfragments. This method involves (a) constructing a replicable expressionvector comprising a first gene encoding an receptor to be mutated, asecond gene encoding at least a portion of a natural or wild-type phagecoat protein wherein the first and second genes are heterologous, and atranscription regulatory element operably linked to the first and secondgenes, thereby forming a gene fusion encoding a fusion protein; (b)mutating the vector at one or more selected positions within the firstgene thereby forming a family of related plasmids; (c) transformingsuitable host cells with the plasmids; (d) infecting the transformedhost cells with a helper phage having a gene encoding the phage coatprotein; (e) culturing the transformed infected host cells underconditions suitable for forming recombinant phagemid particlescontaining at least a portion of the plasmid and capable of transformingthe host, the conditions adjusted so that no more than a minor amount ofphagemid particles display more than one copy of the fusion protein onthe surface of the particle; (f) contacting the phagemid particles witha suitable antigen so that at least a portion of the phagemid particlesbind to the antigen; and (g) separating the phagemid particles that bindfrom those that do not. Steps (d) through (g) can be repeated one ormore times. Preferably in this method the plasmid is under tight controlof the transcription regulatory element, and the culturing conditionsare adjusted so that the amount or number of phagemid particlesdisplaying more than one copy of the fusion protein on the surface ofthe particle is less than about 1%. Also, preferably, the amount ofphagemid particles displaying more than one copy of the fusion proteinis less than 10% of the amount of phagemid particles displaying a singlecopy of the fusion protein. Most preferably, the amount is less than20%. Typically in this method, the expression vector will furthercontain a secretory signal sequence fused to the DNA encoding eachsubunit of the polypeptide and the transcription regulatory element willbe a promoter system. Preferred promoter systems are selected from lacZ, λ_(PL), tac, T7 polymerase, tryptophan, and alkaline phosphatasepromoters and combinations thereof. Also, normally the method willemploy a helper phage selected from M13K07, M13R408, M13-VCS, and Phi X174. The preferred helper phage is M13K07, and the preferred coatprotein is the M13 Phage gene III coat protein. The preferred host is E.coli, and protease-deficient strains of E. coli.

Further details of the foregoing and similar mutagenesis techniques arefound in general textbooks, such as, for example, Sambrook et al.,supra, and Current Protocols in Molecular Biology, Ausubel et al eds.,supra.

Naturally-occurring amino acids are divided into groups based on commonside chain properties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophobic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gin, his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic: trp, tyr, phe.

Conservative substitutions involve exchanging a member within one groupfor another member within the same group, whereas non-conservativesubstitutions will entail exchanging a member of one of these classesfor another. Variants obtained by non-conservative substitutions areexpected to result in significant changes in the biologicalproperties/function of the obtained variant, and may result in TRAFvariants which block TNF biological activities, especially if they areexclusively or primarily mediated by TNF-R2. Amino acid positions thatare conserved among various species and/or various receptors of the TRAFfamily are generally substituted in a relatively conservative manner ifthe goal is to retain biological function.

Amino acid sequence deletions generally range from about 1 to 30residues, more preferably about 1 to 10 residues, and typically arecontiguous. Deletions may be introduced into regions not directlyinvolved in the interaction with the TNF-R2 intracellular domain.

Amino acid insertions include amino- and/or carboxyl-terminal fusionsranging in length from one residue to polypeptides containing a hundredor more residues, as well as intrasequence insertions of single ormultiple amino acid residues. Intrasequence insertions (i.e. insertionswithin the TRAF protein amino acid sequence) may range generally fromabout 1 to 10 residues, more preferably 1 to 5 residues, more preferably1 to 3 residues. Examples of terminal insertions include the TRAFpolypeptides with an N-terminal methionyl residue, an artifact of itsdirect expression in bacterial recombinant cell culture, and fusion of aheterologous N-terminal signal sequence to the N-terminus of the TRAFmolecule to facilitate the secretion of the mature TRAF from recombinanthost cells. Such signal sequences will generally be obtained from, andthus homologous to, the intended host cell species. Suitable sequencesinclude STII or Ipp for E. coli, alpha factor for yeast, and viralsignals such as herpes gD for mammalian cells.

Other insertional variants of the native TRAF molecules include thefusion of the N- or C-terminus of the TRAF molecule to immunogenicpolypeptides, e.g. bacterial polypeptides such as beta-lactamase or anenzyme encoded by the E. coli trp locus, or yeast protein, andC-terminal fusions with proteins having a long half-life such asimmunoglobulin regions (preferably immunoglobulin constant regions),albumin, or ferritin, as described in WO 89/02922 published on 6 Apr.1989.

Since it is often difficult to predict in advance the characteristics ofa variant TRAF, it will be appreciated that some screening will beneeded to select the optimum variant.

3. Insertion of DNA into a Cloning Vehicle

Once the nucleic acid encoding a native or variant TRAF is available, itis generally ligated into a replicable expression vector for furthercloning (amplification of the DNA), or for expression.

Expression and cloning vectors are well known in the art and contain anucleic acid sequence that enables the vector to replicate in one ormore selected host cells. The selection of the appropriate vector willdepend on 1) whether it is to be used for DNA amplification or for DNAexpression, 2) the size of the DNA to be inserted into the vector, and3) the host cell to be transformed with the vector. Each vector containsvarious components depending on its function (amplification of DNA ofexpression of DNA) and the host cell for which it is compatible. Thevector components generally include, but are not limited to, one or moreof the following: a signal sequence, an origin of replication, one ormore marker genes, an enhancer element, a promoter, and a transcriptiontermination sequence.

(i) Signal Sequence Component

In general, the signal sequence may be a component of the vector, or itmay be a part of the TRAF molecule that is inserted into the vector. Ifthe signal sequence is heterologous, it should be selected such that itis recognized and processed (i.e. cleaved by a signal peptidase) by thehost cell.

As the TRAF molecules are intracellular proteins, they are unlikely tohave a native signal sequence. Heterologous signal sequences suitablefor prokaryotic host cells are prokaryotic signal sequences, such as thealkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin IIleaders. For yeast secretion the yeast invertase, alpha factor, or acidphosphatase leaders may be used. In mammalian cell expression mammaliansignal sequences are suitable.

(ii) Origin of Replication Component

Both expression and cloning vectors contain a nucleic acid sequence thatenabled the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomes, and includesorigins of replication or autonomously replicating sequences. Suchsequence are well known for a variety of bacteria, yeast and viruses.The origin of replication from the well-known plasmid pBR322 is suitablefor most gram negative bacteria, the 2 μ plasmid origin for yeast andvarious viral origins (SV40, polyoma, adenovirus, VSV or BPV) are usefulfor cloning vectors in mammalian cells. Origins of replication are notneeded for mammalian expression vectors (the SV40 origin may typicallybe used only because it contains the early promoter). Most expressionvectors are "shuttle" vectors, i.e. they are capable of replication inat least one class of organisms but can be transfected into anotherorganism for expression. For example, a vector is cloned in E. coli andthen the same vector is transfected into yeast or mammalian cells forexpression even though it is not capable of replicating independently ofthe host cell chromosome.

DNA is also cloned by insertion into the host genome. This is readilyaccomplished using Bacillus species as hosts, for example, by includingin the vector a DNA sequence that is complementary to a sequence foundin Bacillus genomic DNA. Transfection of Bacillus with this vectorresults in homologous recombination with the genome and insertion of theDNA encoding the desired heterologous polypeptide. However, the recoveryof genomic DNA is more complex than that of an exogenously replicatedvector because restriction enzyme digestion is required to excise theencoded polypeptide molecule.

(iii) Selection Gene Component

Expression and cloning vectors should contain a selection gene, alsotermed a selectable marker. This is a gene that encodes a proteinnecessary for the survival or growth of a host cell transformed with thevector. The presence of this gene ensures that any host cell whichdeletes the vector will not obtain an advantage in growth orreproduction over transformed hosts. Typical selection genes encodeproteins that (a) confer resistance to antibiotics or other toxins, e.g.ampicillin, neomycin, methotrexate or tetracycline, (b) complementauxotrophic deficiencies, or (c) supply critical nutrients not availablefrom complex media, e.g. the gene encoding D-alanine racemase forbacilli.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene express a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin Southern et al, J. Molec. Appl. Genet. 1, 327(1982)!, mycophenolic acid Mulligan et al., Science 209, 1422 (1980)!,or hygromycin Sudgen et al, Mol. Cel. Biol. 5, 410-413 (1985)!. Thethree examples given above employ bacterial genes under eukaryoticcontrol to convey resistance to the appropriate drug G418 or neomycin(geneticin), xgpt (mycophenolic acid), or hygromycin, respectively.

Other examples of suitable selectable markers for mammalian cells aredihydrofolate reductase (DHFR) or thymidine kinase. Such markers enablethe identification of cells which were competent to take up the desirednucleic acid. The mammalian cell transformants are placed underselection pressure which only the transformants are uniquely adapted tosurvive by virtue of having taken up the marker. Selection pressure isimposed by culturing the transformants under conditions in which theconcentration of selection agent in the medium is successively changed,thereby leading to amplification of both the selection gene and the DNAthat encodes the desired polypeptide. Amplification is the process bywhich genes in greater demand for the production of a protein criticalfor growth are reiterated in tandem within the chromosomes of successivegenerations of recombinant cells. Increased quantities of the desiredpolypeptide are synthesized from the amplified DNA.

For example, cells transformed with the DHFR selection gene are firstidentified by culturing all of the transformants in a culture mediumwhich lacks hypoxanthine, glycine, and thymidine. An appropriate hostcell in this case is the Chinese hamster ovary (CHO) cell line deficientin DHFR activity, prepared and propagated as described by Urlaub andChasin, Proc. Nat'l. Acad. Sci. USA 77, 4216 (1980). A particularlyuseful DHFR is a mutant DHFR that is highly resistant to MTX (EP117,060). This selection agent can be used with any otherwise suitablehost, e.g. ATCC No. CCL61 CHO-K1, notwithstanding the presence ofendogenous DHFR. The DNA encoding DHFR and the desired polypeptide,respectively, then is amplified by exposure to an agent (methotrexate,or MTX) that inactivates the DHFR. One ensures that the cell requiresmore DHFR (and consequently amplifies all exogenous DNA) by selectingonly for cells that can grow in successive rounds of ever-greater MTXconcentration. Alternatively, hosts co-transformed with genes encodingthe desired polypeptide, wild-type DHFR, and another selectable markersuch as the neo gene can be identified using a selection agent for theselectable marker such as G418 and then selected and amplified usingmethotrexate in a wild-type host that contains endogenous DHFR. (Seealso U.S. Pat. No. 4,965,199).

A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid YRp7 (Stinchcomb et al, 1979, Nature 282:39; Kingsmanet al., 1979, Gene 7: 141; or Tschemper et al., 1980, Gene 10:157). Thetrp1 gene provides a selection marker for a mutant strain of yeastlacking the ability to grow in tryptophan, for example, ATCC No. 44076or PEP4-1 (Jones, 1977, Genetics 85:12). The presence of the trpl lesionin the yeast host cell genome then provides an effective environment fordetecting transformation by growth in the absence of tryptophan.Similarly, Leu2 deficient yeast strains (ATCC 20,622 or 38,626) arecomplemented by known plasmids bearing the Leu2 gene.

(iv) Promoter Component

Expression vectors, unlike cloning vectors, should contain a promoterwhich is recognized by the host organism and is operably linked to thenucleic acid encoding the desired polypeptide. Promoters areuntranslated sequences located upstream from the start codon of astructural gene (generally within about 100 to 1000 bp) that control thetranscription and translation of nucleic acid under their control. Theytypically fall into two classes, inducible and constitutive. Induciblepromoters are promoters that initiate increased levels of transcriptionfrom DNA under their control in response to some change in cultureconditions, e.g. the presence or absence of a nutrient or a change intemperature. At this time a large number of promoters recognized by avariety of potential host cells are well known. These promoters areoperably linked to DNA encoding the desired polypeptide by removing themfrom their gene of origin by restriction enzyme digestion, followed byinsertion 5' to the start codon for the polypeptide to be expressed.This is not to say that the genomic promoter for a TRAF polypeptide isnot usable. However, heterologous promoters generally will result ingreater transcription and higher yields of expressed TRAFs as comparedto the native TRAF promoters.

Promoters suitable for use with prokaryotic hosts include theβ-Iactamase and lactose promoter systems (Chang et al., Nature 275:615(1978); and Goeddel et al., Nature 281:544 (1979)), alkalinephosphatase, a tryptophan (trp) promoter system (Goeddel, Nucleic AcidsRes. 8:4057 (1980) and EPO Appln. Publ. No. 36,776) and hybrid promoterssuch as the tac promoter (H. de Boer et al., Proc. Nat'l. Acad. Sci. USA80:21-25 (1983)). However, other known bacterial promoters are suitable.Their nucleotide sequences have been published, thereby enabling askilled worker operably to ligate them to DNA encoding TRAF (Siebenlistet al., Cell 20:269 (1980)) using linkers or adaptors to supply anyrequired restriction sites. Promoters for use in bacterial systems alsowill contain a Shine-Dalgarno (S. D.) sequence operably linked to theDNA encoding a TRAF.

Suitable promoting sequences for use with yeast hosts include thepromoters for 3-phosphoglycerate kinase (Hitzeman et al. J. Biol. Chem.255:2073 (1980)) or other glycolytic enzymes (Hess et al., J. Adv.Enzyme Reg. 7:149 (1978); and Holland, Biochemistry 17:4900 (1978)),such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase,pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphateisomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphateisomerase, phosphoglucose isomerase, and glucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, met allothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin R. Hitzeman et al, EP 73,657A. Yeast enhancers also areadvantageously used with yeast promoters.

Promoter sequences are known for eukaryotes. Virtually all eukaryoticgenes have an AT-rich region located approximately 25 to 30 basesupstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CXCAAT region where X may be any nucleotide. At the3' end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3' end of the codingsequence. All of these sequences are suitably inserted into mammalianexpression vectors.

TRAF transcription from vectors in mammalian host cells may becontrolled by promoters obtained from the genomes of viruses such aspolyoma virus, fowlpox virus (UK 2,211,504 published 5 Jul. 1989),adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcomavirus, cytomegalovirus, a retrovirus, hepatitisoB virus and mostpreferably Simian Virus 40 (SV40), from heterologous mammalianpromoters, e.g. the actin promoter or an immunoglobulin promoter, fromheat shock promoters, and from the promoter normally associated with theTRAF sequence, provided such promoters are compatible with the host cellsystems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment which also contains the SV40 viralorigin of replication Fiers et al., Nature 273:113 (1978), Mulligan andBerg, Science 209, 1422-1427 (1980); Pavlakis et al., Proc. Natl. Acad.Sci. USA 78, 7398-7402 (1981)!. The immediate early promoter of thehuman cytomegalovirus is conveniently obtained as a HindIII Erestriction fragment Greenaway et al., Gene 18, 355-360 (1982)!. Asystem for expressing DNA in mammalian hosts using the bovine papillomavirus as a vector is disclosed in U.S. Pat. No. 4,419,446. Amodification of this system is described in U.S. Pat. No. 4,601,978. Seealso, Gray et al, Nature 295, 503-508 (1982) on expressing cDNA encodinghuman immune interferon in monkey cells; Reyes et al., Nature 297,598-601 (1982) on expressing human β-interferon cDNA in mouse cellsunder the control of a thymidine kinase promoter from herpes simplexvirus; Canaani and Berg,

Proc. Natl. Acad. Sci. USA 79, 5166-5170 (1982) on expression of thehuman interferon β1 gene in cultured mouse and rabbit cells; and Gormanet al., Proc. Natl. Acad. Sci., USA 79, 6777-6781 (1982) on expressionof bacterial CAT sequences in CV-1 monkey kidney cells, chicken embryofibroblasts, Chinese hamster ovary cells, HeLa cells, and mouse HIN-3T3cells using the Rous sarcoma virus long terminal repeat as a promoter.

(v) Enhancer Element Component

Transcription of a DNA encoding the TRAFs of the present invention byhigher eukaryotes is often increased by inserting an enhancer sequenceinto the vector. Enhancers are cis-acting elements of DNA, usually aboutfrom 10 to 300 bp, that act on a promoter to increase its transcription.Enhancers are relatively orientation and position independent havingbeen found 5' Laimins et al, Proc. Natl. Acad. Sci. USA 78, 993 (1981)!and 3' Lasky et al, Mol Cel. Biol. 3, 1108 (1983)! to the transcriptionunit, within an intron Banedi et al., Cell 33, 729 (1983)! as well aswithin the coding sequence itself Osborne et al., Mol. Cel. Biol. 4,1293 (1984)!. Many enhancer sequences are now known from mammalian genes(goblin, elactase, albumin, α-fetoprotein and insulin). Typically,however, one will use an enhabncer from a eukaryotic cell virus.Examples include the SV40 enhancer on the late side of the replicationorigin (bp 100-270), the cytomegalovirus early promoter enhancer, thepolyoma enhancer on the late side of the replication origin, andadenovirus enhancers. See also Yaniv, Nature 297, 17-18 (1982) onenhancing elements for activation of eukaryotic promoters. The enhancermay be spliced into the vector at a position 5' or 3' to the TRAF DNA,but is preferably located at a site 5' from the promoter.

(vi) Transcription Termination Component

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5' and, occasionally 3' untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding the TRAF. The 3' untranslated regions alsoinclude transcription termination sites.

Construction of suitable vectors containing one or more of the abovelisted components, the desired coding and control sequences, employsstandard ligation techniques. Isolated plasmids or DNA fragments arecleaved, tailored, and religated in the form desired to generate theplasmids required.

For analysis to confirm correct sequences in plasmids constructed, theligation mixtures are used to transform E. coli K12 strain 294 (ATCC31,446) and successful transformants selected by ampicillin ortetracycline resistance where appropriate. Plasmids from thetransformants are prepared, analyzed by restriction endonucleasedigestion, and/or sequenced by the method of Messing et al, NucleicAcids Res. 9, 309 (1981) or by the method of Maxam et al, Methods inEnzymology 65, 499 (1980).

Particularly useful in the practice of this invention are expressionvectors that provide for the transient expression in mammalian cells ofDNA encoding a TRAF. In general, transient expression involves the useof an expression vector that is able to replicate efficiently in a hostcell, such that the host cell accumulates many copies of the expressionvector and, in turn, synthesizes high levels of a desired polypeptideencoded by the expression vector. Transient systems, comprising asuitable expression vector and a host cell, allow for the convenientpositive identification of polypeptides encoded by clones DNAs, as wellas for the rapid screening of such polypeptides for desired biologicalor physiological properties. Thus, transient expression systems areparticularly useful in the invention for purposes of identifying analogsand variants of a TRAF.

Other methods, vectors, and host cells suitable for adaptation to thesynthesis of the TRAF polypeptides in recombinant vertebrate cellculture are described in Getting et al., Nature 293, 620-625 (1981);Mantel et al, Nature 281, 40-46 (1979); Levinson et al; EP 117,060 andEP 117,058. A particularly useful plasmid for mammalian cell cultureexpression of the TRAF polypeptides is pRK5 (EP 307,247).

(vii) Construction and Analysis of Vectors

Construction of suitable vectors containing one or more of the abovelisted components employs standard ligation techniques. Isolatedplasmids or DNA fragments are cleaved, tailored, and religated in theform desired to generate the plasmids required.

For analysis to confirm correct sequences in plasmids constructed, theligation mixtures are used to transform E. coli K12 strain 294 (ATCC31,446) and successful transformants selected by ampicillin ortetracycline resistance where appropriate. Plasmids from thetransformants are prepared, analyzed by restriction endonucleasedigestion, and/or sequences by the methods of Messing et al., NucleiAcids Res. 9, 309 (1981) or by the method of Maxam et al., Methods inEnzymology 65, 499 (1980).

(viii) Transient Expression Vectors

Particularly useful in the practice of this invention are expressionvectors that provide for the transient expression in mammalian cells ofDNA encoding a TRAF polypeptide. In general, transient expressioninvolves the use of an expression vector that is able to replicateefficiently in a host cell, such that the host cell accumulates manycopies of the expression vector and, in turn, synthesizes high level ofa desired polypeptide encoded by the expression vector. Sambrook et al,supra, pp. 16.17-16.22. Transient expression systems, comprising asuitable expression vector and a host cell, allow for the convenientpositive screening of such polypeptides for desired biological orphysiological properties. Thus transient expression systems areparticularly useful in the invention for purposes of identifying analogsand variants of native TRAF polypeptides with TRAF biological activity.

(ix) Suitable Exemplary Vertebrate Cell Vectors

Other methods, vectors, and host cells suitable for adaptation to thesynthesis of a TRAF polypeptide (including functional derivatives ofnative proteins) in recombinant vertebrate cell culture are described inGething et al., Nature 293, 620-625 (1981); Mantel et al, Nature 281,40-46 (1979); Levinson et al., EP 117,060; and EP 117,058. Aparticularly useful plasmid for mammalian cell culture expression of aTRAF polypeptide is pRK5 (EP 307,247) or pSVI6B (PCT Publication No. WO91/08291).

D. Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the vectors herein are theprokaryote, yeast or higher eukaryote cells described above. Suitableprokaryotes include gram negative or gram positive organisms, forexample E. coli or bacilli. A preferred cloning host is E. coli 294(ATCC 31,446) although other gram negative or gram positive prokaryotessuch as E. coli B, E. coli X1776 (ATCC 31,537), E. coli W3110 (ATCC27,325), Pseudomonas species, or Serratia Marcesans are suitable.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable hosts for vectors herein. Saccharomycescerevisiae, or common baker's yeast, is the most commonly used amonglower eukaryotic host microorganisms. However, a number of other genera,species and strains are commonly available and useful herein, such as S.pombe Beach and Nurse, Nature .290, 140 (1981)!, Kluyveromyces lactisLouvencourt et al, J. Bacteriol. 737 (1983)!; yarrowia (EP 402,226);Pichia pastoris (EP 183,070), Trichoderma reesia (EP 244,234),Neurospora crassa Case et al, Proc. Natl. Acad. Sci. USA 76, 5259-5263(1979)!; and Aspergillus hosts such as A. nidulans Ballance et al,Biochem. Biophys. Res. Commun. 112, 284-289 (1983); Tilburn et al., Gene26, 205-221 (1983); Yelton et al, Proc. Natl. Acad. Sci. USA 81,1470-1474 (1984)! and A. niger Kelly and Hynes, EMBO J. 4, 475-479(1985)!.

Suitable host cells may also derive from multicellular organisms. Suchhost cells are capable of complex processing and glycosylationactivities. In principle, any higher eukaryotic cell culture isworkable, whether from vertebrate or invertebrate culture, althoughcells from mammals such as humans are preferred. Examples ofinvertebrate cells include plants and insect cells. Numerous baculoviralstrains and variants and corresponding permissive insect host cells fromhosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti(mosquito), Aedes albopictus (mosquito), Drosophila melangaster(fruitfly), and Bombyx mori host cells have been identified. See, e.g.Luckow et al., Bio/Technology 6, 47-55 (1988); Miller et al., in GeneticEngineering, Setlow, J. K. et al, eds., Vol. 8 (Plenum Publishing,1986), pp. 277-279; and Maeda et al, Nature 315, 592-594 (1985). Avariety of such viral strains are publicly available, e.g. the L-1variant of Autographa californica NPV, and such viruses may be used asthe virus herein according to the present invention, particularly fortransfection of Spodoptera frugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,and tobacco can be utilized as hosts. Typically, plant cells aretransfected by incubation with certain strains of the bacteriumAgrobacterium tumefaciens, which has been previously manipulated tocontain the TRAF DNA. During incubation of the plant cell culture withA. tumefaciens, the DNA encoding a TRAF is transferred to the plant cellhost such that it is transfected, and will, under appropriateconditions, express the TRAF DNA. In addition, regulatory and signalsequences compatible with plant cells are available, such as thenopaline synthase promoter and polyadenylation signal sequences.Depicker et al, J. Mol. Appl. Gen. 1, 561 (1982). In addition, DNAsegments isolated from the upstream region of the T-DNA 780 gene arecapable of activating or increasing transcription levels ofplant-expressible genes in recombinant DNA-containing plant tissue. SeeEP 321,196 published 21 Jun. 1989.

However, interest has been greatest in vertebrate cells, and propagationof vertebrate cells in culture (tissue culture) is per se well known.See Tissue Culture, Academic Press, Kruse and Patterson, editors (1973).Examples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cellline 293 or 293 cells subcloned for growth in suspension culture, Grahamet al, J. Gen. Virol. 36, 59 (1977)!; baby hamster kidney cells 9BHK,ATCC CCL 10); Chinese hamster ovary cells/-DHFR CHO, Urlaub and Chasin,Proc. Natl. Acad. Sci. USA 77, 4216 (1980)!; mouse sertolli cells TM4,Mather, Biol. Reprod. 23, 243-251 (1980)!; monkey kidney cells (CV1 ATCCCCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587);human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells(MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442);human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells Mather etal, Annals N.Y. Acad. Sci. 383, 44068 (1982)!; MRC 5 cells; FS4 cells;and a human hepatoma cell line (Hep G2). Preferred host cells are humanembryonic kidney 293 and Chinese hamster ovary cells.

Particularly preferred host cells for the purpose of the presentinvention are vertebrate cells producing the TRAF polypeptides.

Host cells are transfected and preferably transformed with theabove-described expression or cloning vectors and cultured inconventional nutrient media modified as is appropriate for inducingpromoters or selecting transformants containing amplified genes.

E. Culturing the Host Cells

Prokaryotes cells used to produced the TRAF polypeptides of thisinvention are cultured in suitable media as describe generally inSambrook et al., supra.

Mammalian cells can be cultured in a variety of media. Commerciallyavailable media such as Ham's F10 (Sigma), Minimal Essential Medium(MEM, Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium(DMEM, Sigma) are suitable for culturing the host cells. In addition,any of the media described in Ham and Wallace, Meth. Enzymol. 58, 44(1979); Barnes and Sato, Anal. Biochem. 102, 255 (1980), U.S. Pat. Nos.4,767,704; 4,657,866; 4,927,762; or 4,560,655; WO 90/03430; WO 87/00195or U.S. Pat. No. Re. 30,985 may be used as culture media for the hostcells. Any of these media may be supplemented as necessary with hormonesand/or other growth factors (such as insulin, transferrin, or epidermalgrowth factor), salts (such as sodium chloride, calcium, magnesium, andphosphate), buffers (such as HEPES), nucleosides (such as adenosine andthymidine), antibiotics (such as Gentamycin™ drug) trace elements(defined as inorganic compounds usually present at final concentrationsin the micromolar range), and glucose or an equivalent energy source.Any other necessary supplements may also be included at appropriateconcentrations that would be known to those skilled in the art. Theculture conditions, such as temperature, pH and the like, suitably arethose previously used with the host cell selected for cloning orexpression, as the case may be, and will be apparent to the ordinaryartisan.

The host cells referred to in this disclosure encompass cells in invitro cell culture as well as cells that are within a host animal orplant.

It is further envisioned that the TRAF polypeptides of this inventionmay be produced by homologous recombination, or with recombinantproduction methods utilizing control elements introduced into cellsalready containing DNA encoding the particular TRAF.

F. Detecting Gene Amplification/Expression

Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantirate the transcription of mRNA Thomas, Proc. Natl.Acad. Sci. USA 77, 5201-5205 (1980)!, dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Various labels may be employed, most commonlyradioisotopes, particularly ³² P. However, other techniques may also beemployed, such as using biotin-modified nucleotides for introductioninto a polynucleotide. The biotin then serves as a site for binding toavidin or antibodies, which may be labeled with a wide variety oflabels, such as radionuclides, fluorescers, enzymes, or the like.Alternatively, antibodies may be employed that can recognize specificduplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybridduplexes or DNA-protein duplexes. The antibodies in turn may be labeledand the assay may be carried out where the duplex is bound to thesurface, so that upon the formation of duplex on the surface, thepresence of antibody bound to the duplex can be detected.

Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of tissue sections andassay of cell culture or body fluids, to quantitate directly theexpression of gene product. With immunohistochemical stainingtechniques, a cell sample is prepared, typically by dehydration andfixation, followed by reaction with labeled antibodies specific for thegene product coupled, where the labels are usually visually detectable,such as enzymatic labels, fluorescent labels, luminescent labels, andthe like. A particularly sensitive staining technique suitable for usein the present invention is described by Hse et al, Am. J. Clin. Pharm.75, 734-738 (1980).

Antibodies useful for immunohistochemical staining and/or assay ofsample fluids may be either monoclonal or polyclonal, and may beprepared in any animal. Conveniently, the antibodies may be preparedagainst a native TRAF polypeptide, or against a synthetic peptide basedon the DNA sequence provided herein as described further hereinbelow.

G. Purification of the TRAF Polypeptides

The TRAF polypeptide is typically recovered from host cell lysates.

When the TRAF polypeptide is expressed in a recombinant cell other thanone of human origin, the TRAF is completely free of proteins orpolypeptides of human origin. However, it is necessary to purify theTRAF protein from recombinant cell proteins or polypeptides to obtainpreparations that are substantially homogenous as to the TRAF. As afirst step, the culture medium or lysate is centrifuged to removeparticulate cell debris. The membrane and soluble protein fractions arethen separated. The TRAF protein may then be purified from the solubleprotein fraction. The following procedures are exemplary of suitablepurification procedures: fractionation on immunoaffinity or ion-exchangecolumns; ethanol precipitation; reverse phase HPLC; chromatography onsilica or on a cation exchange resin such as DEAE; chromatofocusing;SDS-PAGE; ammonium sulfate precipitation; gel filtration using, forexample, Sephadex G-75; and protein A Sepharose columns to removecontaminants such as IgG. Specific purification procedures have beendescribed hereinabove.

TRAF functional derivatives in which residues have been deleted,inserted and/or substituted are recovered in the same fashion as thenative receptor chains, taking into account of any substantial changesin properties occasioned by the alteration. For example, fusion of theTRAF protein with another protein or polypeptide, e.g. a bacterial orviral antigen, facilitates purification; an immunoaffinity columncontaining antibody to the antigen can be used to absorb the fusion.Immunoaffinity columns such as a rabbit polyclonal anti-TRAF column canbe employed to absorb TRAF variant by binding to at least one remainingimmune epitope. A protease inhibitor, such as phenyl methyl sulfonylfluoride (PMSF) also may be useful to inhibit proteolytic degradationduring purification, and antibiotics may be included to prevent thegrowth of adventitious contaminants. The TRAF proteins of the presentinvention are conveniently purified by affinity chromatography, basedupon their ability to specifically associate with the intracellulardomain of a TNF-R2.

One skilled in the art will appreciate that purification methodssuitable for native TRAF may require modification to account for changesin the character of a native TRAF or its variants upon expression inrecombinant cell culture.

H. Covalent Modifications of TRAF Polypeptides

Covalent modifications of TRAF are included within the scope herein.Such modifications are traditionally introduced by reacting targetedamino acid residues of the TRAF with an organic derivatizing agent thatis capable of reacting with selected sides or terminal residues, or byharnessing mechanisms of post-translational modifications that functionin selected recombinant host cells. The resultant covalent derivativesare useful in programs directed at identifying residues important forbiological activity, for immunoassaVs of the TRAF, or for thepreparation of anti-TRAF receptor antibodies for immunoaffinitypurification of the recombinant. For example, complete inactivation ofthe biological activity of the protein after reaction with ninhydrinwould suggest that at least one arginyl or lysyl residue is critical forits activity, whereafter the individual residues which were modifiedunder the conditions selected are identified by isolation of a peptidefragment containing the modified amino acid residue. Such modificationsare within the ordinary skill in the art and are performed without undueexperimentation.

Cysteinyl residues most commonly are reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesalso are derivatized by reaction with bromotrifluoroacetone,α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing α-amino-containing residues includeimidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylisourea; 2,4-pentanedione; and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pK_(a) of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues may be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidizole and tetranitromethane are used to formO-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosylresidues are iodinated using ¹²⁵ I or ¹³¹ I to prepare labeled proteinsfor use in radioimmunoassay.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R'--N═C═N--R') such as1-cyclohexyl-3-(2-morpholinyl-4-ethyl) carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore,aspartyl and glutamyl residues are converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues falls within the scope of this invention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl, threonyl or tyrosylresidues, methylation of the a-amino groups of lysine, arginine, andhistidine side chains (T. E. Creighton, Proteins: Structure andMolecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-861983!), acetylation of the N-terminal amine, and amidation of anyC-terminal carboxyl group. The molecules may further be covalentlylinked to nonproteinaceous polymers, e.g. polyethylene glycol,polypropylene glycol or polyoxyalkylenes, in the manner set forth inU.S. Ser. No. 07/275,296 or U.S. Pat. Nos. 4,640,835; 4,496,689;4,301,144; 4,670,417; 4,791,192 or 4,179,337.

Derivatization with bifunctional agents is useful for preparingintramolecular aggregates of the TRAF with polypeptides as well as forcross-linking the TRAF polypeptide to a water insoluble support matrixor surface for use in assays or affinity purification. In addition, astudy of interchain cross-links will provide direct information onconformational structure. Commonly used cross-linking agents include1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, homobifunctional imidoesters, andbifunctional maleimides. Derivatizing agents such as methyl-3-(p-azidophenyl)dithio!propioimidateyield photoactivatable intermediateswhich are capable of forming cross-links in the presence of light.Alternatively, reactive water insoluble matrices such as cyanogenbromide activated carbohydrates and the systems reactive substratesdescribed in U.S. Pat. Nos. 3,959,642; 3,969,287; 3,691,016; 4,195,128;4,247,642; 4,229,537; 4,055,635; and 4,330,440 are employed for proteinimmobilization and cross-linking.

Certain post-translational modifications are the result of the action ofrecombinant host cells on the expressed polypeptide. Glutaminyl andaspariginyl residues are frequently post-translationally deamidated tothe corresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues falls within the scope of this invention.

Other post-translational modifications include hydroxylation of prolineand lysine, phosphorylation of hydroxyl groups of seryl, threonyl ortyrosyl residues, methylation of the a-amino groups of lysine, arginine,and histidine side chains T.E. Creighton, Proteins: Structure andMolecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86(1983)!.

Other derivatives comprise the novel peptides of this inventioncovalently bonded to a nonproteinaceous polymer. The nonproteinaceouspolymer ordinarily is a hydrophilic synthetic polymer, i.e. a polymernot otherwise found in nature. However, polymers which exist in natureand are produced by recombinant or in vitro methods are useful, as arepolymers which are isolated from nature. Hydrophilic polyvinyl polymersfall within the scope of this invention, e.g. polyvinylalcohol andpolyvinylpyrrolidone. Particularly useful are polyvinylalkylene etherssuch a polyethylene glycol, polypropylene glycol.

The TRAF polypeptides may be linked to various nonproteinaceouspolymers, such as polyethylene glycol, polypropylene glycol orpolyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

The TRAF may be entrapped in microcapsules prepared, for example, bycoacervation techniques or by interfacial polymerization, in colloidaldrug delivery systems (e.g. liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules), or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences,16th Edition, Osol, A., Ed. (1980).

I. Glycosylation Variants of the TRAFs

The native TRAFs are believed to be unglycosylated, however, variantshaving glycosylation are within the scope herein. For ease, changes inthe glycosylation pattern of a native polypeptide are usually made atthe DNA level, essentially using the techniques discussed hereinabovewith respect to the amino acid sequence variants. Thus, glycosylationsignals can be introduced into the DNA sequence of native TRAFpolypeptides.

Chemical or enzymatic coupling of glycosides to the TRAF molecules ofthe molecules of the present invention may also be used to addcarbohydrate substituents. These procedures are advantageous in thatthey do not require production of the polypeptide that is capable ofO-linked (or N-linked) glycosylation. Depending on the coupling modeused, the sugar(s) may be attached to (a) arginine and histidine, (b)free carboxyl groups, (c) free hydroxyl groups such as those ofcysteine, (d) free sulfhydryl groups such as those of serine, threonine,or hydroxyproline, (e) aromatic residues such as those of phenylalanine,tyrosine, or tryptophan or (f) the amide group of glutamine. Thesemethods are described in WO 87/05330 (published 11 September 1987), andin Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306.

J. Anti-TRAF antibody preparation

(i) Polyclonal antibodies

Polyclonal antibodies to a TRAF molecule generally are raised in animalsby multiple subcutaneous (sc) or intraperitoneal (ip) injections of theTRAF and an adjuvant. It may be useful to conjugate the TRAF or afragment containing the target amino acid sequence to a protein that isimmunogenic in the species to be immunized, e.g. keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsininhibitor using a bifunctional or derivatizing agent, for examplemaleimidobenzoyl sulfosuccinimide ester (conjugation through cysteineresidues), N-hydroxysuccinimide (through lysine residues),glytaraldehyde, succinic anhydride, SOCl₂, or R¹ N═C═NR, where R and R¹are different alkyl groups.

Animals are immunized against the immunogenic conjugates or derivativesby combining 1 mg or 1 μg of conjugate (for rabbits or mice,respectively) with 3 volumes of Freud's complete adjuvant and injectingthe solution intradermally at multiple sites. One month later theanimals are boosted with 1/5 to 1/10 the original amount of conjugate inFreud's complete adjuvant by subcutaneous injection at multiple sites. 7to 14 days later the animals are bled and the serum is assayed foranti-TRAF antibody titer. Animals are boosted until the titer plateaus.Preferably, the animal boosted with the conjugate of the same TRAF, butconjugated to a different protein and/or through a differentcross-linking reagent. Conjugates also can be made in recombinant cellculture as protein fusions. Also, aggregating agents such as alum areused to enhance the immune response.

(ii) Monoclonal antibodies

Monoclonal antibodies are obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally-occurringmutations that may be present in minor amounts. Thus, the modifier"monoclonal" indicates the character of the antibody as not being amixture of discrete antibodies.

For example, the anti-TRAF monoclonal antibodies of the invention may bemade using the hybridoma method first described by Kohler & Milstein,Nature 256:495 (1975), or may be made by recombinant DNA methodsCablily, et al., U.S. Pat. No. 4,816,567!.

In the hybridoma method, a mouse or other appropriate host animal, suchas hamster is immunized as hereinabove described to elicit lymphocytesthat produce or are capable of producing antibodies that willspecifically bind to the protein used for immunization. Alternatively,lymphocytes may be immunized in vitro. Lymphocytes then are fused withmyeloma cells using a suitable fusing agent, such as polyethyleneglycol, to form a hybridoma cell Goding, Monoclonal Antibodies:Principles and Practice, pp.59-103 (Academic Press, 1986)!.

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh level expression of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such as thosederived from MOPC-21 and MPC-1 mouse tumors available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA, and SP-2cells available from the American Type Culture Collection, Rockville,Md. USA. Human myeloma and mouse-human heteromyeloma cell lines alsohave been described for the production of human monoclonal antibodiesKozbor, J. Immunol. 133:3001 (1984); Brodeur, et al, Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987)!.

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against TRAF. Preferably,the binding specificity of monoclonal antibodies produced by hybridomacells is determined by immunoprecipitation or by an in vitro bindingassay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbentassay (ELISA).

The binding affinity of the monoclonal antibody can, for example, bedetermined by the Scatchard analysis of Munson & Pollard, Anal. Biochem.107:220 (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods.Goding, Monoclonal Antibodies: Principles and Practice, pp.59-104(Academic Press, 1986). Suitable culture media for this purpose include,for example, Dulbecco's Modified Eagle's Medium or RPMI-1640 medium. Inaddition, the hybridoma cells may be grown in vivo as ascites tumors inan animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

DNA encoding the monoclonal antibodies of the invention is readilyisolated and sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of murine antibodies). The hybridomacells of the invention serve as a preferred source of such DNA. Onceisolated, the DNA may be placed into expression vectors, which are thentransfected into host cells such as simian COS cells, Chinese hamsterovary (CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of monoclonal antibodiesin the recombinant host cells. The DNA also may be modified, forexample, by substituting the coding sequence for human heavy and lightchain constant domains in place of the homologous murine sequences,Morrison, et al, Proc. Nat. Acad. Sci. 81, 6851 (1984), or by covalentlyjoining to the immunoglobulin coding sequence all or part of the codingsequence for a non-immunoglobulin polypeptide. In that manner,"chimeric" or "hybrid" antibodies are prepared that have the bindingspecificity of an anti-TRAF monoclonal antibody herein.

Typically such non-immunoglobulin polypeptides are substituted for theconstant domains of an antibody of the invention, or they aresubstituted for the variable domains of one antigen-combining site of anantibody of the invention to create a chimeric bivalent antibodycomprising one antigert-combining site having specificity for a TRAF andanother antigen-combining site having specificity for a differentantigen.

Chimeric or hybrid antibodies also may be prepared in vitro using knownmethods in synthetic protein chemistry, including those involvingcrosslinking agents. For example, immunotoxins may be constructed usinga disulfide exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

For diagnostic applications, the antibodies of the invention typicallywill be labeled with a detectable moiety. The detectable moiety can beany one which is capable of producing, either directly or indirectly, adetectable signal. For example, the detectable moiety may be aradioisotope, such as ³ H, ¹⁴ C, ³² P, ³⁵ S, or ¹²⁵ I, a fluorescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin; biotin; radioactive isotopic labels, such as,e.g., ¹²⁵ I, ³² P, ¹⁴ C, or ³ H, or an enzyme, such as alkalinephosphatase, beta-galactosidase or horseradish peroxidase.

Any method known in the art for separately conjugating the antibody tothe detectable moiety may be employed, including those methods describedby Hunter, et al, Nature 144:945 (1962); David, et al, Biochemistry13:1014 (1974); Pain, et al, J. Immunol. Meth. 40:219 (1981); andNygren, J. Histochem. and Cytochem. 30:407 (1982).

The antibodies of the present invention may be employed in any knownassay method, such as competitive binding assays, direct and indirectsandwich assays, and immunoprecipitation assays. Zola, MonoclonalAntibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc., 1987).

Competitive binding assays rely on the ability of a labeled standard(which may be a TRAF polypeptide or an immunologically reactive portionthereof) to compete with the test sample analyte (TRAF) for binding witha limited amount of antibody. The amount of TRAF in the test sample isinversely proportional to the amount of standard that becomes bound tothe antibodies. To facilitate determining the amount of standard thatbecomes bound, the antibodies generally are insolubilized before orafter the competition, so that the standard and analyte that are boundto the antibodies may conveniently be separated from the standard andanalyte which remain unbound.

Sandwich assays involve the use of two antibodies, each capable ofbinding to a different immunogenic portion, or epitope, of the proteinto be detected. In a sandwich assay, the test sample analyte is bound bya first antibody which is immobilized on a solid support, and thereaftera second antibody binds to the analyte, thus forming an insoluble threepart complex. David & Greene, U.S. Pat. No. 4,376,110. The secondantibody may itself be labeled with a detectable moiety (direct sandwichassays) or may be measured using an anti-immunoglobulin antibody that islabeled with a detectable moiety (indirect sandwich assay). For example,one type of sandwich assay is an ELISA assay, in which case thedetectable moiety is an enzyme.

(iii) Humanized antibodies

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as "import" residues, whichare typically taken from an "import" variable domain. Humanization canbe essentially performed following the method of Winter and co-workersJones et al, Nature 321, 522-525 (1986); Riechmann et al, Nature 332,323-327 (1988); Verhoeyen et al., Science 239, 1534-1536 (1988)!, bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such "humanized" antibodiesare chimeric antibodies (Cablily, supra), wherein substantially lessthan an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

It is important that antibodies be humanized with retention of highaffinity for the antigen and other favorable biological properties. Toachieve this goal, according to a preferred method, humanized antibodiesare prepared by a process of analysis of the parental sequences andvarious conceptual humanized products using three dimensional models ofthe parental and humanized sequences. Three dimensional immunoglobulinmodels are commonly available and are familiar to those skilled in theart. Computer programs are available which illustrate and displayprobable three-dimensional conformational structures of selectedcandidate immunoglobulin sequences. Inspection of these displays permitsanalysis of the likely role of the residues in the functioning of thecandidate immunoglobulin sequence, i.e. the analysis of residues thatinfluence the ability of the candidate immunoglobulin to bind itsantigen. In this way, FR residues can be selected and combined from theconsensus and import sequence so that the desired antibodycharacteristic, such as increased affinity for the target antigen(s), isachieved. In general, the CDR residues are directly and mostsubstantially involved in influencing antigen binding. For furtherdetails see U.S. application Ser. No. 07/934,373 filed 21 Aug. 1992,which is a continuation-in-part of application Ser. No. 07/715,272 filed14 June 1991.

Alternatively, it is now possible to produce transgenic animals (e.g.mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy chain joining region (JH) genein chimeric and germ-line mutant mice results in complete inhibition ofendogenous antibody production. Transfer of the human germ-lineimmunoglobulin gene array in such germ-line mutant mice will result inthe production of human antibodies upon antigen challenge. See, e.g.Jakobovits et al, Proc. Natl. Acad. Sci. USA 90, 2551-255 (1993);Jakobovits et al., Nature 362, 255-258 (1993).

(iv) Bispecific antibodies

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is for aTRAF, the other one is for any other antigen, and preferably for anotherreceptor or receptor subunit. For example, bispecific antibodiesspecifically binding two different TRAFs, or a TNF receptor (preferablyTNF-R2) and a TRAF, are within the scope of the present invention.

Methods for making bispecific antibodies are known in the art.

Traditionally, the recombinant production of bispecific antibodies isbased on the coexpression of two immunoglobulin heavy chain-light chainpairs, where the two heavy chains have different specificities(Millstein and Cuello, Nature 305, 537-539 (1983)). Because of therandom assortment of immunoglobulin heavy and light chains, thesehybridomas (quadromas) produce a potential mixture of 10 differentantibody molecules, of which only one has the correct bispecificstructure. The purification of the correct molecule, which is usuallydone by affinity chromatography steps, is rather cumbersome, and theproduct yields are low. Similar procedures are disclosed in PCTapplication publication No. WO 93/08829 (published 13 May 1993), and inTraunecker et al., EMBO 10, 3655-3659 (1991).

According to a different and more preferred approach, antibody variabledomains with the desired binding specificities (antibody-antigencombining sites) are fused to immunoglobulin constant domain sequences.The fusion preferably is with an immunoglobulin heavy chain constantdomain, comprising at least part of the hinge, and second and thirdconstant regions of an immunoglobulin heavy chain (CH2 and CH3). It ispreferred to have the first heavy chain constant region (CH1) containingthe site necessary for light chain binding, present in at least one ofthe fusions. DNAs encoding the immunoglobulin heavy chain fusions and,if desired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are cotransfected into a suitable host organism.This provides for great flexibility in adjusting the mutual proportionsof the three polypeptide fragments in embodiments when unequal ratios ofthe three polypeptide chains used in the construction provide theoptimum yields. It is, however, possible to insert the coding sequencesfor two or all three polypeptide chains in one expression vector whenthe expression of at least two polypeptide chains in equal ratiosresults in high yields or when the ratios are of no particularsignificance. In a preferred embodiment of this approach, the bispecificantibodies are composed of a hybrid immunoglobulin heavy chain with afirst binding specificity in one arm, and a hybrid immunoglobulin heavychain-light chain pair (providing a second binding specificity) in theother arm. It was found that this asymmetric structure facilitates theseparation of the desired bispecific compound from unwantedimmunoglobulin chain combinations, as the presence of an immunoglobulinlight chain in only one half of the bispecific molecule provides for afacile way of separation. This approach is disclosed in copendingapplication Ser. No. 07/931,811 filed 17 Aug. 1992.

For further details of generating bispecific antibodies see, forexample, Suresh et al., Methods in Enzymology 121, 210 (1986).

(v) Heteroconjugate antibodies

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells (U.S. Pat. No. 4,676,980),and for treatment of HIV infection (PCT application publication Nos. WO91/00360 and WO 92/200373; EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

L. Use of TRAF molecules

Based upon their ability to specifically associate with theintracellular domain of TNF-R2, the TRAF molecules of the presentinvention can be used to purify TNF-R2, which, in turn, is useful in thetreatment of various pathological conditions associated with theexpression of TNF, such as endotoxic (septic) shock and rheumatoidarthritis (RA). The dose regimen effective in the treatment of these andother diseases can be determined by routine experimentation.

Therapeutic formulations of the present invention are prepared forstorage by mixing the active ingredient having the desired degree ofpurity with optional physiologically acceptable carriers, excipients orstabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A.Ed. (1980)), in the form of lyophilized formulations or aqueoussolutions. Acceptable carriers, excipients or stabilizers are nontoxicto recipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate and other organic acids; antioxidantsincluding ascorbic acid; low molecular weight (less than about 10residues) polypeptides; proteins, such as serum albumin, gelatin orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone,amino acids such as glycine, glutamine, asparagine, arginine or lysine;monosaccharides, disaccharides and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugaralcohols such as mannitol or sorbitol; salt-forming counterions such assodium; and/or nonionic surfactants such as Tween, Pluronics or PEG.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively), in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, supra.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes, prior to or following lyophilization and reconstitution.

Therapeutic compositions herein generally are placed into a containerhaving a sterile access port, for example, an intravenous solution bagor vial having a stopper pierceable by a hypodermic injection needle.

The route of administration is in accord with known methods, e.g.injection or infusion by intravenous, intraperitoneal, intracerebral,intramuscular, intraocular, intraarterial or intralesional routes,topical administration, or by sustained release systems.

Suitable examples of sustained release preparations includesemipermeable polymer matrices in the form of shaped articles, e.g.films, or microcapsules. Sustained release matrices include polyesters,hydrogels, polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymersof L-glutamic acid and gamma ethyI-L-glutamate (U. Sidman et al., 1983,"Biopolymers" 22 (1): 547-556), poly 2-hydroxyethyl-methacrylate) (R.Langer, et al., 1981, "J. Biomed. Mater. Res." 15: 167-277 and R.Langer, 1982, "Chem. Tech." 12: 98-105), ethylene vinyl acetate (R.Langer et al., Id.) or poly-D-(-)-3-hydroxybutyric acid (EP 133,988A).Sustained release compositions also include liposomes. Liposomescontaining a molecule within the scope of the present invention areprepared by methods known per se: DE 3,218,121A; Epstein et al., 1985,"Proc. Natl. Acad. Sci. U.S.A" 82: 3688-3692; Hwang et el., 1980, "Proc.Natl, Acad. Sci. U.S.A" 77: 4030-4034; EP 52322A; EP 36676A; EP 88046A;EP 143949A; EP 142641 A; Japanese patent application 83-118008; U.S.Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324A. Ordinarily theliposomes are of the small (about 200-800 Angsttoms) unilamelar type inwhich the lipid content is greater than about 30 mol. % cholesterol, theselected proportion being adjusted for the optimal NT-4 therapy.

An effective amount of a molecule of the present invention to beemployed therapeutically will depend, for example, upon the therapeuticobjectives, the route of administration, and the condition of thepatient. Accordingly, it will be necessary for the therapist to titerthe dosage and modify the route of administration as required to obtainthe optimal therapeutic effect. A typical daily dosage might range fromabout 1 μg/kg to up to 100 mg/kg or more, depending on the factorsmentioned above. Typically, the clinician will administer a molecule ofthe present invention until a dosage is reached that provides therequired biological effect. The progress of this therapy is easilymonitored by conventional assays.

TRAF molecules may additionally be used to generate blocking(antagonist) or agonist anti-TRAF antibodies, which can be used to blockor mimic TNF biological activities mediated (exclusively or partially)by TNF-R2, or to purify other TRAF proteins having an epitope to whichthe antibodies bind. Methods for generating anti-TRAF antibodies havebeen described hereinabove. Other TRAF proteins may be identified andpurified, for example, by using the "two-hybrid" assay or its modifiedforms. Thus, factors (including native TRAF proteins and theirfunctional derivatives) that interact with the intracellular domain ofTNF-R2 primarily by dimerizing with a TRAF directly binding to thatdomain (e.g. TRAF2) can be identified by expressing nucleic acidmolecules encoding two fusion proteins in a single host cell transfectedwith nucleic acid encoding a TRAF capable of specific binding the TNF-R2intracellular domain. Specifically, nucleic acid molecules encoding afirst polypeptide comprising a fusion of an intracellular domainsequence of a native TNF-R2 to the DNA-binding domain of atranscriptional activator, and a second polypeptide comprising a fusionof a candidate polypeptide factor to the activation domain of atranscriptional activator are expressed in a single host celltransfected with nucleic acid encoding a polypeptide factor capable ofstrong specific binding to the intracellular domain of TNF-R2 (e.g.TRAF2), and with nucleic acid encoding a reporter gene. The associationof the candidate polypeptide with the intracellular domain of TNF-R2 orwith the polypeptide factor capable of binding to the intracellulardomain of TNF-R2 is monitored by detecting the polypeptide encoded bythe reporter gene.

TRAF molecules (including native TRAF polypeptides and functionalderivatives) can further be used in commercial screening assays toidentify further molecules that inhibit TNF signalling by disrupting theassociation of such TRAF molecules with TNF-R2. Such screening assaysmay, for example, be performed in a two-hybrid assay format as discussedhereinabove.

Further details of the invention will be apparent from the followingnon-limiting examples.

EXAMPLE 1 Purification of TRAF-1

A. Cell Culture and Biological Reagents

The murine interleukin 2 (IL-2)-dependent cytotoxic T cell line CT6(Ranges et al. J. Immunol. 142, 1203-1208 1989!) was cultured in RPMI1640 media supplemented with 10-20 units recombinant human IL-2(Boehringer Mannhelm), 10-15% fet al calf serum (Hyclone), 2 mML-glutamine, 10⁻⁵ M β-mercaptoethanol, 100 units of penicillin per ml,and 100 μg of streptomycin per ml (GIBCO/BRL). The human T-cell lymphomaline Jurkat was obtained from the American Type Culture Collection(ATCC; Rockville, Md.) and maintained in RPMI 1640 media containing 10%fetal calf serum. The human embryonic kidney cell line 293 (ATCC CRL1573) and 293 cells overexpressing the hTNF-R2 (293/TNF-R2) weremaintained as described (Pennica et al., J. Biol. Chem. 267, 21172-211781992!). Recombinant hTNF and recombinant mTNF (specific activity of >10⁷units/mg) were provided by the Genentech Manufacturing Group. The rabbitanti-human and anti-murine TNF-R2 antibodies have been describedpreviously (Pennica et al., supra; Tartaglia et al., J. Biol. Chem. 267,4304-4307 1991!). Anti-human TNF-R1 monoclonal antibody 986 (IgG2aisotype) and anti-human TNF-R2 monoclonal antibodies 1036, 1035 and 1038(IgG2b, IgG2a and IgG2b isotypes, respectively) were produced asdescribed (Pennica et al., Biochemistry 31, 1134-1141 1992!).

B. Mutational Analysis of the Intracellular Domain of hTNF-R2

It has been shown that the TNF induced proliferation of murine CT6 cellsis mediated by the 75 kd TNF receptor (TNF-R2; Tartaglia et al., 1991,supra). In addition, TNF-R2 activates the transcription factor NF-KB(Lenardo & Baltimore, Cell 58: 227-229 1989!) and mediates thetranscriptional induction of the granulocyte-macrophage colonystimulating factor (GM-CSF) gene (Miyatake et al., EMBO J. 4, 2561-25681985!; Stanley et al, EMBO J. 4, 2569-2573 1985!) and the A20 zincfinger protein gene (Opipari et al, J. Biol. Chem. 265, 14705-147081990!) in CT6 cells (FIG. 1).

To identify sequences within the intracellular domain of the hTNF-R2(hTNF-R2icd) that are required for TNF signaling a series of mutanthTNF-R2 expression vectors was generated that encode receptors withtruncated intracellular domains. DNA fragments containing C-terminallytruncated hTNR-R2icds were amplified from the full length expressionvector pRK-TNF-R2 (Tartaglia et al., Cell 73, 213-216 1993!) by PCR withPfu DNA polymerase (Stratagene). PCR was run for 20 cycles (45 s at 95°C.; 60 s at 55° C.; 60 s at 72° C.) after an initial step of 6 min at95° C. A 0.5 kb DNA fragment encoding an intracellular domain whichlacks amino acids 424-439 of the wild type hTNF-R2 was amplified usingthe oligonucleotide primers 5'-CCTTGTGCCTGCAGAGAGAAG-3' (SEQ. ID. NO:23) and 5'-CTAGGTTAACTTTCGGTGCTCCCCAGCAGGGTCTC-3' (SEQ. ID. NO. 24). Thefragment was digested with Pstl and Hindll, gel purified, and re-clonedinto the hTNF-R2 cDNA using the expression vector pRIS (Tartaglia &Goeddel, J. Biol. Chem. 267, 4304-4307 1992!; hTNF-R2(-16)). Similarmutant hTNF-R2 expression vectors were generated that encode receptorslacking amino acids 403-439 (5'-CTAGGTTAACTGGAGAAGGGGACCTGCTCGTCCTT-3'(SEQ. ID NO: 25); hTNF-R2(-37)), amino acids 381-439(5'-CTAGGTTAACTGCTGGCTTGGGAGGAGCACTGTGA-3' (SEQ. ID NO: 26);hTNF-R2(-59)), amino acids 346-439 (5'-CTAGGTTAACTGCTCCCGGTGCTGGCCCGGGCCTC-3' (SEQ. ID NO: 27); hTNF-R2(-94)) and amino acids 308 439(5'-CTAGGTTAACTGCACTGGCCGAGCTCTCCAGGGA-3' (SEQ. ID NO: 28);hTNF-R2(-132)). A deletion of amino acids 304-345 of hTNF-R2 wasconstructed by partial digest of pRK-TNF-R2 with Sacl and re-ligation ofthe vector (hTNF-R2(α304-345)). A deletion of the entire intracellulardomain of hTNF-R2 was constructed from pRK-TNF-R2 by replacement ofsequences between the Pstl site adjacent to the transmembrane domain andthe Clal site with a double-stranded oligonucleotide(5'-GTGATGAGAATTCAT-3' (SEQ. ID NO: 29) and 5'-CGATGAATTCTCATCACTGCA-3') (SEQ. ID NO: 30) containing an in-frame stop codonimmediately following Gln²⁷³ (hTNF-R2(-166)). A mutation convertingSer³⁹³ into Ala was introduced into the hTNF-R2 cDNA by site-directedmutagenesis as described (Tartaglia et al, Cell 74, 845-853 1993!;hTNF-R2(S393A)). Verification of correctly modified cDNAs was determinedby double-strand sequencing using the Sequenase 2.0 Sequencing Kit (U.S.Biochemical).

The expression vectors encoding the intact and truncated hTNF-R2 wereintroduced into CT6 cells by electropotation. 5×10⁶ cells in 0.3 ml RPMI1640 media were cotransfected with 0.5 μg of Scal-digested pRK.neo(Tartaglia & Goeddel, 1992, supra) and 20 pg of Scal-digested hTNF-R2expression vector using the Bio-Rad Gene Pulser with CapacitanceExtender (0.4 cm cuvette, 960 μF, 250 V). Electroporated cells wereresuspended in 50 ml media and after 2 days plated into 96-wellmicrotiter plates by limiting dilution in selective media containing 100μg/ml G418 (GIBCO/BRL). After three weeks, individual G418-resistantclones were picked and expanded. Clones that express the hTNF-R2 wereidentified by FACS analysis as described (Table 1; Pennica et al., J.Biol. Chem. 1992, supra).

Proliferation of CT6 clones expressing the full length and truncatedhTNF-R2 was measured by ³ H!thymidine incorporation as described(Tartaglia et al., Proc. Natl. Acad. Sci. USA 88, 9292-9296 1991!).NF-KB activation was analyzed by electrophoretic mobility shift assaywith nuclear extracts prepared from stimulated or unstimulated CT6 cellsas described (Schutze et al., Cell 71,765-776 1992!).

Table 1 shows that the transfected hTNF-R2 signals proliferation andNF-KB activation in CT6 cells. In addition, mutant human receptors whichlack the C-terminal 16 amino acids or the internal 42 amino acids304-345 are still functional in mediating these activities. In contrast,mutant receptors which lack the C-terminal 37 amino acids or containfurther C-terminal deletions are defective in these assays. Theseresults indicate that a region of 78 amino acids within theintracellular domain of hTNF-R2 comprising amino acids 346-423 isrequired for mediating TNF signaling. This region contains a potentialprotein kinase C phosphorylation site (Ser³⁹³ -Pro³⁹⁴ -Lys³⁹⁵) which isconserved in the murine TNF-R2. However, a mutant hTNF-R2 containing Alainstead of Ser.sup. 389 is biologically functional (Table 1) indicatingthat this phosphorylation site is not involved in TNF-R2 mediatedsignaling.

C. Identification of Factors that Associate with the IntracellularDomain of hTNF-R2

To identify factors that are associated with the intracellular domain ofhTNF-R2 immunoprecipitation of the receptor from lysates of ³⁵S!-labeled transfected CT6 cells was performed. 5×10⁶ CT6 cellsexpressing the wild type hTNF-R2 were washed twice with low glucoseDulbecco's modified Eagle's media without cysteine and methionine andincubated in fresh media for 30 min. The cells were seeded into a 100-mmplate in 5 ml media (without cysteine and methionine) containing ³⁵S!cysteine and ³⁵ !methionine (50 μCi of L- ³⁵ S!-in vitro cell labelingmix/ml; Amersham). The cells were incubated for 4 h at 37° C.,stimulated for 10 min with 100 ng/ml hTNF, harvested, washed twice withcold PBS and lysed for 20 min at 4° C. in 1 ml of 0.1% NP40 lysis buffercontaining 50 mM HEPES pH 7.2, 250 mM NaCl, 10% Glycerol, 2 mM EDTA, 1mM PMSF, 1 μg/ml Benzamidine, 1 μg/ml Aprotinin, 1 μg/ml Leupeptin.Nuclear and cell debris were removed by centrifugation at 10,000×g for10 min at 4° C. The cell lysate was precleared for. 1 h at 4° C. with 50μl Pansorbin (Calbiochem). The lysate was incubated for 8 h at 4° C.with 1 μg of each of the anti-hTNF-R2 monoclonal antibodies 1035 and1038 (directed against different epitopes of the extracellular domain ofthe hTNF-R2) that had been preabsorbed with 1 ml of unlabeled lysatefrom untransfected CT6 cells and collected with 15 μl of proteinA-agarose beads (Oncogene Science). The beads were washed extensivelywith lysis buffer, resuspended in SDS sample buffer and the supernatantelectrophoresed on a 4-12% or 8% Tris/glycine polyacrylamide gel. Thegel was fixed, incubated in Amplify (Amersham), dried, and exposed tofilm at -80° C.

Several bands in the range of 45-50 kd and one band of approximately 68kd were specifically coprecipitated with the immunoprecipitated hTNF-R2in CT6 cells (FIG. 2a). The same result was obtained when the hTNF-R2was immunoprecipitated from unlabeled 293/TNF-R2 cells followed byincubation with labeled lysate from untransfected CT6 cells (FIG. 2b).The pattern of bands coprecipitated with hTNF-R2 was identicalregardless of whether the lysate was prepared from cells that had beenstimulated with hTNF or left unstimulated, indicating that theseproteins are constitutively associated with the hTNF-R2. This is similarto results observed for the tyrosine kinase JAK2 which is associatedwith the intracellular domain of the erythropoietin receptor (Witthuhnet al., Cell 74, 227-236 1993!). In order to establish a large scalepurification procedure for factors that associate with the hTNF-R2icd,the intracellular domain of hTNF-R2 was expressed as a glutathioneS-transferase (GST) fusion protein (Smith & Johnson, 1988, supra). Theintracellular domain of hTNF-R2 was amplified from pRK-TNF-R2 by PCRwith Pfu DNA polymerase as described above using the oligonucleotideprimers 5'-GATCGGATCCAAAAAGAAGCC CTTGTGCCTGCA-3' (SEQ. ID NO: 31) and5'-GCCTGGTTAACTGGGC-3' (SEQ. ID NO: 32). The amplified 0.55 kb DNAfragment was blunt-ended, digested with Barnill and cloned intoBarnHI/Sinai-digested pGEX-2TK vector (Pharmacia; pGST-hTNF-R2icd). ThepGST-hTNF-R2icd plasmid was transformed into a protease deficient strainof E. coli K12 carrying the lacl^(q) gene on the chromosome (Genentech),an overnight culture diluted 1:10 in fresh LB-medium containing 100μg/ml carbenicillin and grown at 37° C. for 2 h. After induction with0.1 mM IPTG, cells were grown for 1 h at 37° C., pelleted and washedonce with cold PBS. The cells were resuspended in 1/100 culture volumeof resuspension buffer containing 20 mM Tris HCI pH 7.5, 1M NaCl, 5 mMEDTA, lmM DTT, 1 mM PMSF, 1 μg/ml Benzamidine, 1 μg/ml Aprotinin, 1μg/ml Leupeptin. After sonication on ice, insoluble material was removedby centrifugation at 10,000×g for 15 min at 4° C. Triton X-100 was addedto 1% and the cell lysate incubated for 30 min at room temperature on arotator with 500 μl of a 50% slurry of glutathione-agarose beads(sulphur linkage; Sigma) in PBS per 1 l culture volume. The beads werecollected by brief centrifugation at 500×g and washed extensively withresuspension buffer. An aliquot of the purified GST-hTNF-R2icd fusionprotein was analyzed by SDS-PAGE (FIG. 3). Concentrations of 5-8 mgfusion protein/ml of swollen beads were obtained routinely.

To prepare a covalently linked GST-hTNF-R2icd fusion protein affinitymatrix, the fusion protein was eluted from glutathione-agarose beads bycompetition with free glutathione using 3×30 min washes with 1 beadvolume of 250 mM Tris HCl pH 8.0 containing 50 mM reduced glutathione(Sigma). The eluted fusion protein was dialyzed against 0.1M HEPES pH7.2, 150 mM NaCl and covalently coupled to Affigel 10/15 (2:1 ratio;Bio-Rad) according to the instructions of the manufacturer. Fusionprotein concentrations of up to 10 mg/ml of swollen beads were obtained.Coprecipitation experiments with GST-hTNF-R2icd fusion protein wereperformed by incubating 3 μl fusion protein beads with 1 ml of celllysate prepared from ³⁵ !-labeled CT6 cells as described above. After 8h at 4° C. the fusion protein beads were extensively washed with lysisbuffer and analyzed by SDS-PAGE and autoradiography. A pattern of bandswas found to specifically coprecipitate with the GST-hTNF-R2icd fusionprotein either bound to glutathione-agarose beads or covalently coupledto Affigel 10/15 (FIG. 4) that was very similar in size to the bandscoprecipitating with the immunoprecipitated hTNF-R2 (see FIG. 3). Thissuggests that the GST-hTNF-R2icd fusion protein expressed in E. colidoes associate with the same intracellular factors as the wild typehTNF-R2 in CT6 cells.

Expression vectors were made that encode GST-hTNF-R2icd fusion proteinswith mutant intracellular domains according to the mutational analysisdescribed above. Using the same strategy as for the wild type hTNF-R2icdDNA fragments encoding the mutant -16, -37, -59 and Δ304-345 hTNF-R2intracellular domains were amplified by PCR and cloned into the pGEX-2TKvector. In addition, a 0.14 kb DNA fragment was amplified using theoligonucleotide primers 5'-GATCGGATCCGGAGACACAGATTCCAGCCCC-3' (SEQ IDNO: 53) and 5'-GATCGAATTCTTAACTCTTCGGTGCTCCCCAGCAG-3' (SEQ. ID NO: 54),digested with BamHi and EcoRI and cloned into pGEX-2TK. This DNAfragment encodes a peptide of 41 amino acids that correspond to aminoacids 384-424 of the hTNF-R2icd. The fusion proteins were expressed,purified and assayed for coprecipitating proteins as described above.

As shown in FIG. 5 the GST-hTNF-R2icd fusion proteins containing theintracellular domains of the functional receptor mutants hTNF-R2(--16)and hTNF-R2(Δ304-345) coprecipitated the same bands as the fusionprotein containing the wild type hTNF-R2icd. In contrast, theGST-hTNF-R2icd fusion proteins which contain the intracellular domainsof the inactive mutants hTNF-R2(-37) and hTNF-R2(-59) did notcoprecipitate these bands. This correlation between the biologicalactivity of hTNF-R2s with mutant intracellular domains and thecoprecipitation results obtained with the corresponding GST-hTNF-R2icdfusion proteins supports the observation that the wild typeGST-hTNF-R2icd fusion protein associates with the same intracellularfactors as the immunoprecipitated hTNF-R2.

In addition, the GST-hTNF-R2icd(384-424) fusion protein was able tocoprecipitate the bands at 45-50 kd and 68 kd although to a weakerextent than the other fusion proteins (FIG. 5). The 41 amino acids ofthe hTNF-R2icd contained in this GST-fusion protein are comprised withinthe 78 amino acids region of the hTNF-R2icd that has been identified tobe required for mediating TNF signaling in CT6 cells (see above). Thissuggests that this short region of the hTNF-R2icd is sufficient tomediate the association of potential signaling molecules with thereceptor.

Competition coprecipitation experiments were performed in which thehTNF-R2 was immunoprecipitated from unlabeled 293/TNF-R2 cells and thenincubated with labeled CT6 cell lysate that had been precleared with 50μl of GST-hTNF-R2icd fusion protein beads. Preincubation of the CT6extracts with GST beads alone or GST-hTNF-R2icd(-37) andGST-hTNF-R2icd(-59) fusion protein beads had no effect on the pattern ofproteins coprecipitating with the immunoprecipitated hTNF-R2 (FIG. 6).However, if the cell lysate had been precleared with GST-hTNF-R2icd orGST-hTNF-R2icd(-16) fusion protein beads, these proteins did notcoprecipitate with the immunoprecipitated hTNF-R2 (FIGS. 6A and 6B),indicating that they had been depleted from the labeled CT6 cell extractby the GST-hTNF-R2icd fusion proteins. This result demonstrates that thewild type GST-hTNF-R2icd fusion protein associates with the sameintracellular factors as the immunoprecipitated hTNF-R2. Consequently,this GST-fusion protein material can be used for large scalepurification of factors that are associated with the intracellulardomain the of hTNF-R2.

Coprecipitation experiments of GST-hTNF-R2icd fusion beads with celllysate prepared from ³⁵ S! labeled human Jurkat cells revealed a patternof coprecipitating proteins very similar in size to the pattern observedwith murine CT6 lysates (FIG. 7). This suggests that the TNF-R2associated factors are closely related between the mouse and humanspecies.

To investigate the subcellular localization of the hTNF-R2 associatedfactors, cytoplasmic and membrane fractions from CT6 cells were preparedessentially as described (Deutscher, Methods in Enzymol. 182: AcademicPress, San Diego 1990!). Briefly, ³⁵ S! labeled CT6 cells were washedonce with cold PBS and once with isotonic salt buffer containing 50 mMTris HCl pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 μg/ml Aprotininand 1 μg/ml Leupeptin. Cells were resuspended in 5 ml isotonic saltbuffer, and lysed in a glass douncer (Wheaton) with 20 strokes using the`B` pestle. Large cell debris were removed by centrifugation at 750×gfor 10 min at 4° C. and the supernatant subjected to ultracentrifugationat 100,000×g for 30 min at 4° C. The supernatant which constitutes thecytoplasmic fraction was removed and the pellet resuspended in 50 mMTris HCl pH 7.4, 1 mM EDTA, 1 mM PMSF, 1 μg/ml Aprotinin, 1 μg/mlLeupeptin. This crude membrane fraction was layered on a cushion of 35%w/v sucrose in PBS and centrifuged at 30,000×g for 45 min at 4° C. Thepurified cell membrane fraction at the interface between the sucrose andthe buffer phases was removed carefully, concentrated by centrifugationat 100,000×g for 30 min at 4° C. and extracted with 0.1% NP40 lysisbuffer for 30 min at 4° C. The cell membrane lysate and the cytoplasmicfraction were used in coprecipitation experiments with GST-hTNF-R2fusion protein beads as described above.

The factors associating with the hTNF-R2icd were found to be localizedin the cytoplasmic cell fraction (FIG. 8). A small amount could also bedetected in the purified cell membrane fraction consistent with theobservation that these factors are constitutively associated with thehTNF-R2icd (see above).

D. Large scale purification

For large scale purification of hTNF-R2icd associated factors 60 I ofCT6 cells (3×10¹⁰ cells) were harvested and washed twice with cold PBS.All subsequent operations were carried out at 4° C. The cells were lysedby adding 120 ml of 0. 1% NP40 lysis buffer containing 100 mM NaCl androcked gently for 30 min. Insoluble material was removed bycentrifugation for 10 min at 10,000×g. The supernatant was thencentrifuged at 100,000×g for 1 hr and dialyzed against lysis buffercontaining 500 mM NaCl. All subsequent purification steps were carriedout in lysis buffer containing 500 mM NaCl. The cell lysate was passedthrough a 15 ml glutathione-agarose GST-hTNF-R2icd(-37) fusion proteinpreabsorption column. The flow-through was applied to a 0.3 ml Affigel10/15 GST-hTNF-R2icd fusion protein affinity column. For control, thelysate was run through a similar Affigel 10/15 GST-hTNF-R2icd(-37)fusion protein affinity column in parallel. After extensive washing,proteins bound to the resins were eluted with five column volumes ofImmunoPure Gentle AglAb Elution Buffer (Pierce) containing, 0.1M DTT,precipitated with Methanol/Chloroform and resuspended in SDS samplebuffer containing 5% SDS. One tenth of the material was separated bySDS-PAGE under reducing conditions and visualized by silver staining(FIG. 9). Protein bands that were specifically eluted from theGST-hTNF-R2icd fusion protein affinity column were observed atapproximately 45-50 kd and 68-70 kd.

The remaining purified material was separated by SDS-PAGE,electrophoretically transferred to PVDF sequencing membrane (Millipore)and proteins visualized by staining with R250. The protein band at 45-50kd (TNF Receptor Associated Factor 1 or TRAF1) was cut out and subjectedto amino acid sequence analysis by automated Edman degradation on anApplied Biosystems sequencer. Since the material proved to beN-terminally blocked, internal sequence information was obtained fromindividual peptides that were purified by reversed phase capillary HPLCafter protease digestion prior to sequence analysis. Two peptides thatwere obtained from trypsin and lysine C digestion, respectively, had thesequences APMALER and KHAYVK (SEQ>ID. NOS: 41 and 42).

EXAMPLE 2 Recombinant production of TRAF-1

The following degenerate oligonucleotides were designed based on thesequences of the above peptides: BP50-1 sense, 5'-GCNCCNATGGCNYTNGARC/AG(SEQ. ID. NOs: 33-35); BP50-1 antisense, 5'-CT/GYTCNARNGCCATNGGNGC (SEQ.ID NOs: 36-38); BP50-11 sense, 5'-AARCAYGCNTAY GTNAA (SEQ. ID NO: 39);BP50-11 antisense, 5'-TTNACRTANGCRTGYTT (SEQ. ID NO: 40). 1 pg poly(A)⁺mRNA isolated from CT6 cells was oligo(dT)-primed and reversetranscribed using the cDNA Cycle Kit (Invitrogen) according to theinstructions of the manufacturer. First-strand CT6 cDNA was subjected toPCR with combinations of the degenerate oligonucleotides listed aboveusing a Cetus GeneAmp Kit and Perkin-Elmer Thermocycler. The PCR was runfor 35 cycles (45 s at 95° C.; 60 s at 55° C.; 150 s at 72° C.) after aninitial step of 6 min at 95° C. The PCR products were analyzed byelectrophoresis on a 1.6% agarose gel. The PCR reaction obtained withthe primer combination BP50-1 sense and BP50-11 antisense CoomaniiBrilliant Blue R-250 (Sigma) contained an amplified DNA fragment ofapproximately 0.75 kb. This fragment was gel-purified, subcloned intopBluescript KS (Stratagene), and sequenced.

A 0.65 kb Pstl DNA fragment was isolated from the cloned PCR fragmentand labeled with λ-³² P!dCTP using the T7 Quick Prime Kit (Pharmacia).The labeled fragment was used to screen approximately 1×10^(e)recombinant phage clones from a CT6 cDNA library that had beenconstructed in λgt22a using the Supercript Lambda System For cDNASynthesis And λ Cloning (GIBCO/BRL) according to the instructions of themanufacturer. Hybridization and washing of the filters were carried outunder high-stringency conditions according to standard protocols(Ausubel et al, Current Protocols in Molecular Biology, Green PublishingAssociates and Wiley Interscience, New York 1987). Four positive cloneswere plaque-purified by a secondary screen. The cDNA inserts of thesephage clones were subcloned into pBluescript KS and sequenced on bothstrands (FIGS. 10A and 10B). The longest of the cDNAs was found to be 2kb. The other three cDNA clones represented truncated versions of the 2kb cDNA clone. The 2 kb cDNA clone contained an open reading frameencoding a protein of 409 amino acids (FIGS. 10A and 10B). Within thepredicted protein were the sequences APMALER (SEQ. ID. NO: 41) andKHAYVK (SEQ. ID. NO: 42), as well as the sequences PGSNLGS (SEQ. ID. NO:43) and KDDTMFLK (SEQ. ID. NO: 44) which correspond to two other peptidesequences obtained from protein sequence analysis. These results confirmthat the isolated 2 kb cDNA clone encodes the purified TRAF1.

To analyze similarities between TRAF1 and other known sequences, theTRAF1 sequence was searched against the Genentech protein database. Noobvious similarity of significance between TRAF1 and any other knownprotein was found, indicating that TRAF1 is a novel molecule.

EXAMPLE 3 Identification and cloning of TRAF2

To directly isolate genes coding for proteins that associate with theintracellular domain of TNF-R2 the yeast two-hybrid system for thedetection of protein-protein interactions (Fields & Song, Nature 340,245-246 1989!) was used. The intracellular domain of hTNF-R2 wasamplified from pRK-TNF-R2 by PCR with Pfu DNA polymerase as describedabove using the oligonucleotide primers5'-TCGATCGTCGACCAAAAAGAAGCCCTCCTGCCTACAA-3' (SEQ. ID NO: 45) and5'-CTAGAGATCTCAGGGGTCAGGCCACTTT-3' (SEQ. ID. NO: 46). The amplified 0.55kb DNA fragment was digested with Sa/I and Bglll, gel-purified andcloned into the GAL4 DNA-binding domain vector pPC97 (Chevray & Nathans,1992, supra; pPC97-hTNF-R2icd). Similar constructs were made containingthe GAL4 DNA-binding domain fused to the hTNF-R2icd(-16)(5'-CTAGAGATCTGTTAACTTTCGGTGCTCCCCAGCAGGGTCTC-3' (SEQ. ID. NO: 47);pPC97-hTNF-R2icd(-16)), the hTNF-R2icd(-37)(5'-CTAGAGATCTGTTAACTGGAGAAGGGGACCTGCTCGTCC TT-3' (SEQ. ID. NO: 48);pPC97-hTNF-R2icd(-37)), the hTNF-R2icd(-59)(5'-CTAGAGATCTGTTAACTGCTGGCTTGGGAGGAGCACTGTGA-3' (SEQ. ID. NO: 49);pPC97-hTNF-R2icd(-59)) and the intracellular domain of the murine TNF-R2(5'-TCGATCGTCGACCAAAAAGAAGCCCTCCTGCCT ACAA-3' (SEQ. ID NO: 50),5'-CTAGAGATCTCAGGGGTCAGGCCACTTT-3' (SEQ. ID. NO: 51); pPC97-mTNF-R2icd).

A plasmid cDNA library in the GAL4 transcriptional activation domainvector pPC86 (Chevray & Nathans, 1992, supra) was constructed fromSall/Notl-adapted, double-stranded fet al liver stromal cell line 7-4cDNA (a gift of B. Bennett and W. Matthews) as described (Chevray &Nathans, 1992, supra). Plasmid DNA was isolated directly from 2×10⁶transformed E. coli DH10B (GIBCO/BRL) colonies. S. cerevisiae HF7c(CIontech) was sequentially transformed with pPC97-hTNF-R2icd and 250 μglibrary plasmid DNA as described in the Matchmaker Two-Hybrid System(Clontech). The final transformation mixture was plated onto 50 150-mmsynthetic dextrose agar plates lacking L-tryptophan, L-leucine,L-histidine and containing 20 mM 3-aminotriazole (Sigma). A total of2×10⁶ transformed colonies were plated. After 4 days at 30° C. 42surviving His⁺ -colonies were obtained of which 15 were positive in afilter assay for b-galactosidase activity (Breeden & Nasmyth, Regulationof the Yeast HO gene. Cold Spring Harbor Symposia on QuantitativeBiology 50, 643-650, Cold Spring Harbor Laboratory Press, New York,1985). Yeast DNA was prepared (Hoffmann and Winston, Gene 57, 267-2721987!), transformed into E. Coli DH10B by electroporation and coloniescontaining the pPC86 library plasmid identified by restriction analysis.14 out of 15 cDNA inserts had a similar size of approximately 2.1 kb.Restriction analysis with Ddel revealed them to be independent cDNAclones derived from the same mRNA species.

Retransformation of three representative cDNA clones into HF7c cellswith pPC97 and pPC97-hTNF-R2icd, respectively, confirmed that theencoded GAL4 activation domain fusion proteins do not interact with theGAL4 DNA-binding domain alone but only with the GAL4 DNA-bindinghTNF-R2icd fusion protein. The 2.1 kb cDNA insert of one representativeclone (pPC86Y17) was sequenced on both strands (FIGS. 11A and 11B). Inaddition, the 5'- and 3'-regions of 6 other independent cDNA clones weresequenced confirming that they were derived from the same mRNA species.All clones were shown to be fused to the GAL4 DNA-binding domain in thesame reading frame within 20 nucleotides of each other.

Two additional cDNA clones were isolated from a CT6 λ phage cDNA library(see above) and 5 additional clones from a mouse liver λ phage cDNAlibrary (Clontech) using a ³² P!-labeled 0.5 kb Pstl DNA fragment fromthe 5'-region of the pPC86Y17 cDNA insert as hybridization probe. Noneof these cDNA inserts extended the 5'-sequence of the pPC86 cDNAinserts. Furthermore, the size of the pPC86 cDNA inserts correspondsclosely to the size of the actual message as revealed by northern blotanalysis of CT6 mRNA (see below). These findings indicate that the cDNAinserts isolated with the two-hybrid system represent full length clonesand that the fusion to the GAL4 DNA-binding domain occurred in a veryshort 5'-untranslated region in-frame with the initiator ATG at position30 of the pPC86Y17 cDNA insert (see also below). The cDNA clones containan open reading frame encoding a protein of 501 amino acids (TNFReceptor Associated Factor 2 or TRAF2; FIGS. 11A and 11B).

A hornology search of the TRAF2 sequence against the Genentech proteindatabase revealed that TRAF2 is a novel protein containing an N-terminalRING finger sequence motif (Freemont et al., Cell 64, 483-484 1991!;Haupt et al., Cell 65, 753-763 1991!; Inoue et al., supra; FIG. 12a).This sequence motif has been observed in the N-terminal domain of anumber of regulatory proteins and is thought to form two zinc-bindingfinger structures that appear to be involved in protein-DNA interactions(Freemont et al., supra; Haupt et al., supra; Reddy et al., TrendsBiochem. Sci. 17, 344-345 1992!). Members of the RING finger family areputative DNA-binding proteins, some of which are implicated intranscriptional regulation, DNA repair, and site-specific recombination(see FIG. 12A). In addition, the RING finger motif and otherzinc-binding sequence motifs have been discussed to be involved inprotein-protein interactions (Freemont et al., supra; Haupt et al.,supra; Berg, J. Biol. Chem. 265, 6513-6516 1990!). The importance ofthis structural motif in TRAF2 is supported by the finding that all GAL4DNA-binding domain TRAF2 fusions isolated contain the completeN-terminus of TRAF2 (see above). This suggests that the N-terminal RINGfinger domain of TRAF2 is involved in the interaction with theintracellular domain of TNF-R2.

In addition, TRAF2 shares sequence similarity with the zinc finger motifof Xenopus TFIIIA-type zinc finger proteins (Miller et al, EMBO J. 4,1609-1614 1985!; Berg, supra; FIG. 12B). TFIIIA-like zinc finger motifshave also been observed in the RING finger proteins RAD18 and UVS-2(FIG. 12).

No obvious similarity of significance between the C-terminal domain ofTRAF2 and any other known protein was found. A comparison of thesequences of TRAF1 and TRAF2 revealed that they share a high degree ofamino acid identify in their C-terminal domains (53% identity over 230amino acids; FIG. 13). Both proteins constitute members of a new familyof proteins that contain a novel sequence homology motif, the "TRAFdomain". The less conserved N-terminal regions within the TRAF domainsof TRAF1 and TRAF2 can potentially form leucine zipper-like structures(FIGS. 10, 11, 13). The leucine zipper is a α-helical structureoriginally found in a number of DNA-binding proteins that containleucines occurring at intervals of every seventh amino acid (Landschulzet al, Science 240, 1759-1764 1988!; Vinson et al, Science 246, 911-9161989!). This structure mediates protein dimerization by intermolecularinteraction of the leucine side-chains. Leucine zipper structures havealso been predicted for two other RING finger family members, theSS-A/Ro ribonucleoprotein and the gene product of the c-cblproto-oncogene (see FIGS. 12A and 12B). The N-terminal domains of TRAF1and TRAF2 are unrelated, especially with regard to the RING fingerdomain of TRAF2.

EEXAMPLE 4 Functional Analysis of TRAF1 and TRAF2

Hydropathy profiles (Kyte & Doolittle, 1982, Supra) of TRAF1 and TRAF2(FIGS. 14A and 14B) suggest that they lack signal sequences as well asobvious transmembrane regions and are overall hydrophilic. They are thuslikely to represent intracellular proteins which is in accordance withthe cytoplasmic localization of TRAF1 as determined experimentally (seeabove).

Poly(A)⁺ mRNA was prepared from CT6 cells (Badley et al., CurrentOpinion in Structural Biology 3, 11-16, 1988!). Northern analysis(Sambrook et al, 1989, supra) using a radiolabeled TRAF2 hybridizationprobe as described above indicated that TRAF2 is expressed as a 2.1 kbmessage in CT6 cells (FIG. 15A). Similarly, TRAF1 is expressed in CT6cells as a 2 kb message (FIG. 15A).

To examine the tissue distribution of TRAF1 and TRAF2 mRNA, mousemultiple tissue Northern blots (Clontech) were hybridized withradiolabeled TRAF1 and TRAF2 probes according to the instructions of themanufacturer. TRAF2 is expressed constitutively in all mouse tissuesexamined (heart, brain, spleen, lung, liver, skelet al muscle, kidneyand testis; FIG. 15B). The highest expression level was observed inspleen. In contrast, TRAF1 displayed a tissue specific expression. TRAF1mRNA could only be detected in spleen, lung and testis (FIG. 15B).

Cotransformation of pPC86Y17 (pPC86TRAF2) into HF7c cells with the abovedescribed GAL4 DNA-binding TNF-R2icd fusion constructs showed that TRAF2interacts with the wild type intracellular domains of both the human andthe murine TNF-R2 and with the intracellular domain of the biologicallyactive mutant hTNF-R2(-16) (Table 2). However it does not interact withthe GAL4 DNA-binding domain alone nor with the intracellular domains ofthe biologically inactive routants hTNF-R2(-37) and hTNF-R2(-59). Thisis in agreement with the results obtained from coprecipitationexperiments with wild type and mutant GST-hTNF-R2icd fusion proteins inCT6 cell extracts (see above).

An expression vector encoding a GST-TRAF2 fusion protein wasconstructed. The TRAF2 coding region was amplified from pPC86TRAF2 byPCR with Pfu DNA polymerase as described above using the oligonucleotideprimers 5'-GATCGGATCCTTGTGGTGTGTGGGGG TTGT (SEQ. ID. NO: 55) and5'-CCTGGCTGGCCTAATGT (SEQ. ID. NO: 56). The amplified 1.6 kb DNAfragment was blunt-ended using E. coli DNA polymerase I, digested withBarnHi and cloned into BarnHI/Sinai-digested pGEX-2TK vector. TheGST-TRAF2 fusion protein was expressed in the presence of 1 mM ZnCL₂ andpurified as described above. GST and GST-TRAF2 fusion protein beads wereincubated with lysates from 293 and 293FFNF-R2 cells, and analyzed bySDS-PAGE and Western blot analysis (Sambrook et al., "Molecular Cloning:A Laboratory Manual. Cold Spring Harbor Laboratory Press, N.Y. 1989!)using the ECL detection reagent (Amersham). Primary antibodies directedagainst the extracellular domains of hTNF-R2 and hTNF-R1 (as a control)were used at a concentration of 0.5 μg/ml and the secondary sheepanti-mouse horseradish peroxidase conjugate (Amersham) at a dilution of1:6000. As shown in FIG. 16, the GST-TRAF2 fusion protein coprecipitatesthe hTNF-R2 in 293 cell extracts, thus confirming the results obtainedfrom two hybrid analysis.

To test for possible homo- and heteromeric protein-protein interactionsbetween TRAF1, TRAF2 and the intracellular domain of TNF-R2 the 2.1 kbcDNA insert of pPC86TRAF2 was excised by digestion with Sa/I and Notland cloned into pPC97 (pPC97TRAF2). The TRAF1 coding and 3'-untranslatedregion was amplified from the full length TRAF1 cDNA clone inpBluescript KS by PCR with Pfu DNA polymerase as described above usingthe T7 sequencing primer (Stratagene) and the oligonucleotide primer5'-TCGATCGTCGACCGCCTCCAGCTCAGCCCCTGAT-3' (SEQ. ID. NO: 52). Theamplified 1.7 kb DNA fragment was digested with Sa/I and Notl,gel-purified and cloned into both pPC97 and pPC86 (pPC97TRAF1;pPC86TRAF1).

Cotransformation of pPC86TRAF1 into HF7c cells with the GAL4 DNA-bindingTNF-R2 fusion constructs encoding the wild type human and murineintracellular domains indicated that the direct interaction betweenTRAF1 and the intracellular domain of TNF-R2 is weak (Table 2). However,cotransformation of pPC97TRAF1 and pPC86TRAF2 or pPC97TRAF2 andpPC86TRAF 1 revealed that TRAF 1 and TRAF2 interact with each other(Table 2) suggesting that a heterodimeric complex of TRAF1 and TRAF2 isassociated with the intracellular domain of TNF-R2. Subsequently yeastvectors were constructed in which TRAF2 is expressed directly, i. e. notas a GAL4 fusion protein. pPC97TRAF2 was digested with HindIll and Sal/Ito release a 0.5 kb DNA fragment encoding the GAL4 enzyme, gel-purified,and re-ligated (pPCTRAF2). In addition, TRAF2 was amplified frompPC86TRAF2 with Pfu DNA polymerase as described above using theoligonucleotide primers 5'-GATCGACTCGAGATGCCCAAGAAGAAGCGGAAGGTGGCTGCAGCCAGTGTGACTTCCCCT (SEQ. ID. NO: 57) and 5'-CTCTGGCGAAGAAGTCC (SEQ.ID. NO: 58). The amplified 2.1 kb DNA fragment was digested with Xhol,end-filled with Klenow enzyme, digested with Notl, gel-purified andcloned into pPC97 that had been digested with HindIll, end-filled anddigested with Notl (pPCTRAF2NLS). This expression vector encodes thesimian virus 40 large tumor antigert nuclear localization signal(met-pro-lys-lys-lys-arg-lys-val; compare Chevray & Nathans, 1992) fusedto the N-terminus of TRAF2. Transformation of pPCTRAF2NLS but notpPCTRAF2 into HF7c cells harboring the plasmids pPC86TRAF1 andpPC97hTNF-R2icd or pPC97mTNF-R2icd complemented the histidine deficiencyof the host cells (Table 3). This result confirms that a heterodimericcomplex of TRAF1 and TRAF2 interacts with the intracellular domain ofTNF-R2. In this protein complex mainly TRAF2 contacts the receptordirectly potentially through interaction of its RING finger domain withthe C-terminal region of the intracellular domain comprising amino acids304-345 of the human TNF-R2 as suggested from mutational analysis andcoprecipitation experiments (see above). TRAF1 and TRAF2 can also formhomodimeric complexes as shown by cotransformation of pPC97TRAF1 andpPC86TRAF1 or pPC97TRAF2 and pPC86TRAF2 (Table 2). These results suggestthat the homologous C-terminal domains of TRAF1 and TRAF2 represent anovel protein dimerization motif. In analogy, the Coterminal domain ofthe RING finger protein COP1 from Arabidopsis thailane contains a regionwith homology to the β subunit of trimeric G proteins that has beendiscussed to be involved in protein-protein recognition (Deng et el,Cell 71, 791-801 1992!).

Based on the tissue specific expression of TRAF1 (see above), theformation of a heteromeric complex between TRAF1 and TRAF2 can onlyoccur in certain mouse tissues such as spleen, lung and testis. Thisraises the possibility of other TRAF domain proteins as tissue specificdimerization partners for the constitutively expressed TRAF2. Suchtissue specific heterocomplexes with potentially different biologicalactivities could determine different TNF responses mediated by TNF-R2 invarious tissues.

To generate antibodies directed specifically against TRAF1 and TRAF2 theN-terminal domains of both proteins were expressed in E. coli as 6×histag fusions using the QIAexpress system (Qiagen). A DNA fragmentencoding amino acids 2-181 of TRAF1 was amplified from the full lengtheDNA clone in pBluescript KS by PCR with Pfu DNA polymerase as describedabove using the oligonucleotide primers5'-GATCGGATCCGCCTCCAGCTCAGCCCCTGAT (SEQ. ID. NO: 59) and5'-GATCGGATCCAGCCAGCAGCTTCTCCTTCAC (SEQ. ID. NO: 60). The amplified 0.55kb DNA fragment was digested with Barnill and cloned intoBarnill-digested pQE12 vector (Qiagen). Transformants containing thecorrect orientation of the DNA insert in the expression vector weredetermined by restriction analysis (pQETRAF1). Similarly, a DNA fragmentencoding amino acids 1-162 of TRAF2 was amplified from pPC86TRAF2 usingthe oligonucleotide primers 5'-GATCGGATCCTTGTGGTGTGTGGGGGTTGT (SEQ. ID.NO: 61) and 5'-GATCGGATCCGCTCAGG CTC TTTTGGGGCA (SEQ. ID. NO: 62),digested with BamHI and cloned into BamHI-digested pQE12 vector(pQETRAF2). Plasmids pQETRAF1 and pQETRAF2 were transformed into E. coliM15 pREP4! (Qiagen). The cells were grown, induced, harvested, and the6×his tag TRAF1 and TRAF2 fusion proteins purified by Ni-NTA affinitychromatography (Qiagen) under denaturing conditions according to theinstructions of the manufacturer. The purified TRAF1 and TRAF2 fusionproteins were resolved on a 13% Tris/glycine polyacrylamide gel, stainedwith 0.05% Coomassie Brilliant Blue R-250 solution in water, and theappropriate bands excised. Gel slices containing 100-200 μg TRAF1 orTRAF2 fusion protein were used for the immunization of rabbits.

                  TABLE 1                                                         ______________________________________                                        Analysis of CT6 Clones Expressing Human TNF-R2 Mutants                                                .sup.3 H!Thymidine                                             hTNF-R2 Expression                                                                          Incorporation                                                                              NFκB                                CT6 Clone                                                                              (mean fluorescence)                                                                         (fold stimulation)                                                                         Activation                                ______________________________________                                        neo.26   157           0.9          -                                         hR2.30   303           3.4          +                                         hR2.31   350           4.7          +                                          -16.25  464           10.7         +                                          -16.31  465           5.1          +                                          -37.4   478           1.1          -                                          -37.20  439           1.0          -                                          -59.1   276           1.3          -                                          -59.23  374           1.0          -                                          -94.5   515           0.7          -                                          -94.6   477           1.0          -                                         -132.3   296           1.1          -                                         -132.22  344           1.0          -                                         -166.10  361           1.0          -                                         -166.13  318           1.0          -                                         Δ304-345                                                                         469           nd           +                                         S393A.2  407           2.9          nd                                        S393A.8  531           5.5          nd                                        ______________________________________                                    

Expression vectors encoding the intact and truncated hTNF-R2 weretransfected into CT6 cells. The expression levels of wild type or mutantreceptors of individual CT6 clones were analyzed by flow cytometry andvalues are expressed as mean fluorescence. For functional analysis twoindependent CT6 clones were examined for each hTNF-R2 mutant excepthTNF-R2(Δ304-345) which represents a pool of sorted cells. Proliferationwas measured by ³ H!thymidine incorporation of cells that had beentreated for 24 hr with a 1:1000 dilution of anti-hTNF-R2 polyclonalantibody. Values are expressed as fold stimulation compared with cellsthat had been treated with an irrelevant antibody. Data shown are themeans of triplicate determinations. Standard deviations were generallyless than 5%. NF-KB activation was analyzed by electrophoretic mobilityshift assay with nuclear extracts prepared from cells that had beenstimulated for 20 min with a 1:500 dilution of anti-hTNF-R2 polyclonalantibody. A plus sign indicates the induction of NF-KB DNA-bindingactivity compared with nuclear extracts prepared from cells that hadbeen treated with an irrelevant antibody. All CT6 clones retained theability to induce proliferation and NF-KB activation through theendogenous murine TNF-R2 (data not shown). rid, not determined.

                                      TABLE 2                                     __________________________________________________________________________    Interaction between TRAF1, TRAF2 and the Intracellular Domain of TNF-R2       Transformant             Growth on trp--                                                                       Growth on trp--                              DNA-binding domain hybrid                                                                  Activation-domain hybrid                                                                  leu.sup.- medium                                                                      leu.sup.- his.sup.- medium                   __________________________________________________________________________    GAL4(DB)     GAL4(TA)    +       -                                            GAL4(DB)-hTNF-R2icd                                                                        GAL4(TA)    +       -                                            GAL4(DB)-mTNF-R2icd                                                                        GAL4(TA)    +       -                                            GAL4(DB)-hTNF-R2icd(-16)                                                                   GAL4(TA)    +       -                                            GAL4(DB)-hTNF-R2icd(-37)                                                                   GAL4(TA)    +       -                                            GAL4(DB)-hTNF-R2icd(-59)                                                                   GAL4(TA)    +       -                                            GAL4(DB)     GAL4(TA)-TRAF1                                                                            +       -                                            GAL4(DB)     GAL4(TA)-TRAF2                                                                            +       -                                            GAL4(DB)-hTNF-R2icd                                                                        GAL4(TA)-TRAF1                                                                            +       -/+                                          GAL4(DB)-mTNF-R2icd                                                                        GAL4(TA)-TRAF1                                                                            +       -/+                                          GAL4(DB)-hTNF-R2icd                                                                        GAL4(TA)-TRAF2                                                                            +       +                                            GAL4(DB)-mTNF-R2icd                                                                        GAL4(TA)-TRAF2                                                                            +       +                                            GAL4(DB)-hTNF-R2icd(-16)                                                                   GAL4(TA)-TRAF2                                                                            +       +                                            GAL4(DB)-hTNF-R2icd(-37)                                                                   GAL4(TA)-TRAF2                                                                            +       -                                            GAL4(DB)-hTNF-R2icd(-59)                                                                   GAL4(TA)-TRAF2                                                                            +       -                                            GAL4(DB)-TRAF1                                                                             GAL4(TA)    +       -                                            GAL4(DB)-TRAF2                                                                             GAL4(TA)    +       -                                            GAL4(DB)-TRAF1                                                                             GAL4(TA)-TRAF1                                                                            +       +                                            GAL4(DB)-TRAF1                                                                             GAL4(TA)-TRAF2                                                                            +       +                                            GAL4(DB)-TRAF2                                                                             GAL4(TA)-TRAF2                                                                            +       +                                            GAL4(DB)-TRAF2                                                                             GAL4(TA)-TRAF1                                                                            +       +                                            __________________________________________________________________________

HF7c cells were cotransformed with plasmids (see text) encoding variousGAL4 DNA-binding domain (DB) and GAL4 transcriptional activation domain(TA) fusion proteins as indicated. Aliquots of the same transformationmixture were plated onto synthetic dextrose plates lacking trp and leuand plates lacking trp, leu, his and containing 20 mM 3-aminotriazole.Plus signs indicate growth of transformed yeast colonies on therespective plates. Very similar numbers of transformants from the sametransformation mixture (90-100%) were obtained on plates lacking trp,leu, his and on plates lacking trp and leu only. Minus/plus signsindicate that the number of transformants growing on plates lacking trp,leu, his was approximately 1-2% of the number of colonies obtained fromthe same transformation mixture on plates lacking trp and leu only.Filter assays for β-galactosidase activity were performed on coloniesgrowing on plates lacking all three amino acids. All colonies developeda blue color (data not shown).

                  TABLE 3                                                         ______________________________________                                        Interaction between TRAF1, TRAF2 and the Intracellular                        Domain of TNF-R2 (continued)                                                                              Growth on                                         Transformant                trp--                                             DNA-binding  Activation-domain                                                                          Direct    leu.sup.- his.sup.-                       domain hybrid                                                                              hybrid       expression                                                                              medium                                    ______________________________________                                        GAL4(DB)     GAL4(TA)-TRAF1         -                                         GAL4(DB)     GAL4(TA)-TRAF1                                                                             NLS-TRAF2 -                                         GAL4(DB)     GAL4(TA)-TRAF1                                                                             TRAF2     -                                         GAL4(DB)-hTNF-R2icd                                                                        GAL4(TA)-TRAF1         -/+*                                      GAL4(DB)-hTNF-R2icd                                                                        GAL4(TA)-TRAF1                                                                             NLS-TRAF2 +                                         GAL4(DB)-hTNF-R2icd                                                                        GAL4(TA)-TRAF1                                                                             TRAF2     -/+*                                      GAL4(DB)-mTNF-R2icd                                                                        GAL4(TA)-TRAF1         -/+*                                      GAL4(DB)-mTNF-R2icd                                                                        GAL4(TA)-TRAF1                                                                             NLS-TRAF2 +                                         GAL4(DB)-mTNF-R2icd                                                                        GAL4(TA)-TRAF1                                                                             TRAF2     -/+*                                      ______________________________________                                         HF7c cells were sequentially transformed with plasmids (see text) encodin     the indicated GAL4 DNAbinding domain (DB) fusion proteins, the GAL4           transcriptional activation domain (TA) TRAF1 fusion protein, and TRAF2 or     TRAF2 fused to the simian virus 40 large tumor antigen nuclear                localization signal (NLS). The final transformation mixtures were plated      onto synthetic dextrose plates lacking trp, leu, his and containing 20 mM     3aminotriazole. Plus signs indicate growth of transformed yeast colonies      on the respective plates.                                                     *See Table 2.                                                            

While the invention has necessarily been described in conjunction withpreferred embodiments and specific working examples, one of ordinaryskill, after reading the foregoing specification, will be able to effectvarious changes, substitutions or equivalents, and alterations to thesubject matter set forth herein, without departing from the spirit andscope herein. Hence, the invention can be practiced in ways other thanthose specifically described herein. All such modifications are intendedto be within the scope of the present invention.

All references cited herein and the references cited therein are herebyexpressly incorporated by reference.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 62                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2088 bases                                                        (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       CCCAGCCCGGTTCTCTGCCCCAAGGACGCTACCGCCCAATGCGAGCAGAA50                          GGCGGCGCACAGATACAGAAAGTGAGGCTCAGACATATTGAAGACCGTGT100                         GACATAGGGTAGCCAAATGACAGTGTGAGAAAGTGACATTTACTCAAGGC150                         CACCCAGATATCCTGGAGGACCCAGAACCCTGGAGATTCCCATCAGAAAG200                         ACCTTCTGGCCACCTGAAACCCCAAGATGGCCTCCAGCTCAGCCCCTGAT250                         GAAAACGAGTTTCAATTTGGTTGCCCCCCTGCTCCCTGCCAGGACCCATC300                         GGAGCCCAGAGTTCTCTGCTGCACAGCCTGTCTCTCTGAGAACCTGAGAG350                         ATGATGAGGATCGGATCTGTCCTAAATGCAGAGCAGACAACCTCCATCCT400                         GTGAGCCCAGGAAGCCCTCTGACTCAGGAGAAGGTTCACTCTGATGTAGC450                         TGAGGCTGAAATCATGTGCCCCTTTGCAGGTGTTGGCTGTTCCTTCAAGG500                         GGAGCCCACAATCCATGCAGGAGCATGAGGCTACCTCCCAGTCCTCCCAC550                         CTGTACCTGCTGCTGGCGGTCTTAAAGGAGTGGAAATCCTCACCAGGCTC600                         CAACCTAGGGTCTGCACCCATGGCACTGGAGCGGAACCTGTCAGAGCTGC650                         AGCTTCAGGCAGCTGTGGAAGCGACAGGGGACCTGGAGGTAGACTGCTAC700                         CGGGCACCTTGCTGTGAGAGCCAGGAAGAACTGGCCCTGCAGCACTTGGT750                         GAAGGAGAAGCTGCTGGCTCAGCTGGAGGAGAAGCTGCGTGTGTTTGCAA800                         ACATTGTTGCTGTCCTCAACAAGGAAGTGGAGGCTTCCCACCTGGCACTG850                         GCCGCCTCCATCCACCAGAGCCAGTTGGACCGAGAGCACCTCCTGAGCTT900                         GGAGCAGAGGGTGGTGGAATTACAGCAAACCCTGGCTCAAAAAGACCAGG950                         TCCTGGGCAAGCTTGAGCACAGTCTGCGACTCATGGAGGAGGCATCCTTT1000                        GATGGTACTTTCCTGTGGAAGATCACCAATGTCACCAAGCGGTGCCACGA1050                        GTCAGTGTGTGGCCGGACTGTCAGCCTCTTCTCTCCAGCTTTCTACACTG1100                        CCAAGTATGGTTACAAGTTGTGCCTGCGCTTGTACCTGAACGGGGATGGC1150                        TCAGGCAAGAAGACCCACCTGTCCCTCTTCATCGTGATCATGAGAGGAGA1200                        ATACGATGCTCTCCTGCCCTGGCCTTTCAGGAACAAGGTCACCTTTATGC1250                        TACTTGACCAGAACAACCGAGAGCATGCTATTGATGCCTTCCGGCCTGAC1300                        CTGAGCTCAGCCTCCTTCCAGCGGCCACAGAGTGAGACCAACGTGGCCAG1350                        CGGCTGCCCGCTCTTCTTCCCCCTCAGCAAGCTGCAGTCACCCAAGCACG1400                        CCTACGTCAAAGATGACACAATGTTCCTCAAATGCATTGTGGACACTAGT1450                        GCTTAGGGATGGGGGGAGGGGGTGTCTCCTGACAGAACCAGCTTAGACTG1500                        GGGGACTTAGCTAGACAGCCAGGCCCTGCCTGCCCTTGGAGCCCACAGCC1550                        CACGACAAGGAGGAGCCAAGGCTGGCATGACTTCAGCGCCACAGCATGCT1600                        GGTTATGGCTGATGTGAGGCTGGAGAAACGTGTGCGTACAGAGACAGAGT1650                        GGAGGAGAAGACAGAAGTGCTCTTTTCACACAGACTACACGACACCAGGA1700                        GGCCAGCATGCCAGCAGCTTCTGAATGTTGAGACCAGCCTAGATCAGGAT1750                        GAAAAGAGCCAGGCCTGAGGCTTGGACATTGAGCCAAGGCTATGGGGCCT1800                        AAGTGGAGGGGCACTCCTACCAGGACATTCTCTCGAGGTCAGGGCATAAC1850                        TGGAAAAATGCCCCCATCTCTCTGTTCAGACTCAAAACTAGAACCACAGG1900                        GCAGAAGGGTCAGACATTAATGTGAATTTAACCTGCCCTGGACTGAGTTC1950                        CTATGTTAACAGACACGCAAACAGGTAAACCCAGAAACTGCCCTGGGAAA2000                        TGCTTTCTGGCTGCATCTGGAGATCTTTGATGTTTTTACCGACAAAACAA2050                        ATAACAAAAGCCTTGAATTGCAAAAAAAAAAAAAAAAA2088                                    (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 409 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetAlaSerSerSerAlaProAspGluAsnGluPheGlnPheGly                                 151015                                                                        CysProProAlaProCysGlnAspProSerGluProArgValLeu                                 202530                                                                        CysCysThrAlaCysLeuSerGluAsnLeuArgAspAspGluAsp                                 354045                                                                        ArgIleCysProLysCysArgAlaAspAsnLeuHisProValSer                                 505560                                                                        ProGlySerProLeuThrGlnGluLysValHisSerAspValAla                                 657075                                                                        GluAlaGluIleMetCysProPheAlaGlyValGlyCysSerPhe                                 808590                                                                        LysGlySerProGlnSerMetGlnGluHisGluAlaThrSerGln                                 95100105                                                                      SerSerHisLeuTyrLeuLeuLeuAlaValLeuLysGluTrpLys                                 110115120                                                                     SerSerProGlySerAsnLeuGlySerAlaProMetAlaLeuGlu                                 125130135                                                                     ArgAsnLeuSerGluLeuGlnLeuGlnAlaAlaValGluAlaThr                                 140145150                                                                     GlyAspLeuGluValAspCysTyrArgAlaProCysCysGluSer                                 155160165                                                                     GlnGluGluLeuAlaLeuGlnHisLeuValLysGluLysLeuLeu                                 170175180                                                                     AlaGlnLeuGluGluLysLeuArgValPheAlaAsnIleValAla                                 185190195                                                                     ValLeuAsnLysGluValGluAlaSerHisLeuAlaLeuAlaAla                                 200205210                                                                     SerIleHisGlnSerGlnLeuAspArgGluHisLeuLeuSerLeu                                 215220225                                                                     GluGlnArgValValGluLeuGlnGlnThrLeuAlaGlnLysAsp                                 230235240                                                                     GlnValLeuGlyLysLeuGluHisSerLeuArgLeuMetGluGlu                                 245250255                                                                     AlaSerPheAspGlyThrPheLeuTrpLysIleThrAsnValThr                                 260265270                                                                     LysArgCysHisGluSerValCysGlyArgThrValSerLeuPhe                                 275280285                                                                     SerProAlaPheTyrThrAlaLysTyrGlyTyrLysLeuCysLeu                                 290295300                                                                     ArgLeuTyrLeuAsnGlyAspGlySerGlyLysLysThrHisLeu                                 305310315                                                                     SerLeuPheIleValIleMetArgGlyGluTyrAspAlaLeuLeu                                 320325330                                                                     ProTrpProPheArgAsnLysValThrPheMetLeuLeuAspGln                                 335340345                                                                     AsnAsnArgGluHisAlaIleAspAlaPheArgProAspLeuSer                                 350355360                                                                     SerAlaSerPheGlnArgProGlnSerGluThrAsnValAlaSer                                 365370375                                                                     GlyCysProLeuPhePheProLeuSerLysLeuGlnSerProLys                                 380385390                                                                     HisAlaTyrValLysAspAspThrMetPheLeuLysCysIleVal                                 395400405                                                                     AspThrSerAla                                                                  409                                                                           (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2121 bases                                                        (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GCGCGAAGACCGTTGGGGCTTTGTGGTGTGTGGGGGTTGTAACTCACATG50                          GCTGCAGCCAGTGTGACTTCCCCTGGCTCCCTAGAACTGCTACAGCCTGG100                         CTTCTCCAAGACCCTCCTGGGGACCAGGTTAGAAGCCAAGTACCTCTGTT150                         CAGCCTGCAAAAACATCCTGCGGAGGCCTTTCCAGGCCCAGTGTGGGCAC200                         CGCTACTGCTCCTTCTGCCTGACCAGCATCCTCAGCTCTGGGCCCCAGAA250                         CTGTGCTGCCTGTGTCTATGAAGGCCTGTATGAAGAAGGCATTTCTATTT300                         TAGAGAGTAGTTCGGCCTTTCCAGATAACGCTGCCCGCAGAGAGGTGGAG350                         AGCCTGCCAGCTGTCTGTCCCAATGATGGATGCACTTGGAAGGGGACCTT400                         GAAAGAATACGAGAGCTGCCACGAAGGACTTTGCCCATTCCTGCTGACGG450                         AGTGTCCTGCATGTAAAGGCCTGGTCCGCCTCAGCGAGAAGGAGCACCAC500                         ACTGAGCAGGAATGCCCCAAAAGGAGCCTGAGCTGCCAGCACTGCAGAGC550                         ACCCTGTAGCCACGTGGACCTGGAGGTACACTATGAGGTCTGCCCCAAGT600                         TTCCCTTAACCTGTGATGGCTGTGGCAAGAAGAAGATCCCTCGGGAGACG650                         TTTCAGGACCATGTTAGAGCATGCAGCAAATGCCGGGTTCTCTGCAGATT700                         CCACACCGTTGGCTGTTCAGAGATGGTGGAGACTGAGAACCTGCAGGATC750                         ATGAGCTGCAGCGGCTACGGGAACACCTAGCCCTACTGCTGAGCTCATTC800                         TTGGAGGCCCAAGCCTCTCCAGGAACCTTGAACCAGGTGGGGCCAGAGCT850                         ACTCCAGCGGTGCCAGATTTTGGAGCAGAAGATAGCAACCTTTGAGAACA900                         TTGTCTGCGTCTTGAACCGTGAAGTAGAGAGGGTAGCAGTGACTGCAGAG950                         GCTTGTAGCCGGCAGCACCGGCTAGACCAGGACAAGATTGAGGCCCTGAG1000                        TAACAAGGTGCAACAGCTGGAGAGGAGCATCGGCCTCAAGGACCTGGCCA1050                        TGGCTGACCTGGAGCAGAAGGTCTCCGAGTTGGAAGTATCCACCTATGAT1100                        GGGGTCTTCATCTGGAAGATCTCTGACTTCACCAGAAAGCGTCAGGAAGC1150                        CGTAGCTGGCCGGACACCAGCTATCTTCTCCCCAGCCTTCTACACAAGCA1200                        GATATGGCTACAAGATGTGTCTACGAGTCTACTTGAATGGCGACGGCACT1250                        GGGCGGGGAACTCATCTGTCTCTCTTCTTCGTGGTGATGAAAGGCCCCAA1300                        TGATGCTCTGTTGCAGTGGCCTTTTAATCAGAAGGTAACATTGATGTTGC1350                        TGGACCATAACAACCGGGAGCATGTGATCGACGCATTCAGGCCCGATGTA1400                        ACCTCGTCCTCCTTCCAGAGGCCTGTCAGTGACATGAACATCGCCAGTGG1450                        CTGCCCCCTCTTCTGCCCTGTGTCCAAGATGGAGGCCAAGAATTCCTATG1500                        TGCGGGATGATGCGATCTTCATCAAAGCTATTGTGGACCTAACAGGACTC1550                        TAGCCACCCCTGCTAAGAATAGCAGCTCAGTGAGGAGCTGTCACATTAGG1600                        CCAGCCAGGCCCTGCCACACACGGGTGGGCAGGCTTGGTGTAAATGCTGG1650                        GGAGGGCCTCAGCCTAGAGCCAATCACCATCACACAGAAAGGCAGGAAGA1700                        AGCCTCCAGTTGGCCTTCAGCTGGCAAACTGAGTTGGACGGTCCACTGAG1750                        CTCAAGGGCCTGGTGGAGCCCGCTGGGGAGCTTCTCAGCTTTCCAATAGG1800                        AAAGCTCCTGCTGTCTCCTCTGTCTGGGGAAGGGAGAGACCTGTAGGTGG1850                        GTGCTCAGAAAGGGCCTCTCCAGAGAGAGTCTCAAGAGCTGCAGCAGGAG1900                        CAAAGTGACTGGCCTTCCCCACCCCATCCTTTGGAAAAGAGGTAGCGGCT1950                        ACACAGGAGAAGGCATGCGCCTGCAGGGTGTAGCCCAAGAGAGAAGCTCT2000                        CTGAGACATAGGCCCTCACTGGAGAAGGGCCTGCCTGGGCTGCACAGCCT2050                        TGCCAGGTGGCCTGTATGGGGGAGAAGTGATTAAATGTTGAGATGTCACA2100                        CGACAAAAAAAAAAAAAAAAA2121                                                     (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 501 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       MetAlaAlaAlaSerValThrSerProGlySerLeuGluLeuLeu                                 151015                                                                        GlnProGlyPheSerLysThrLeuLeuGlyThrArgLeuGluAla                                 202530                                                                        LysTyrLeuCysSerAlaCysLysAsnIleLeuArgArgProPhe                                 354045                                                                        GlnAlaGlnCysGlyHisArgTyrCysSerPheCysLeuThrSer                                 505560                                                                        IleLeuSerSerGlyProGlnAsnCysAlaAlaCysValTyrGlu                                 657075                                                                        GlyLeuTyrGluGluGlyIleSerIleLeuGluSerSerSerAla                                 808590                                                                        PheProAspAsnAlaAlaArgArgGluValGluSerLeuProAla                                 95100105                                                                      ValCysProAsnAspGlyCysThrTrpLysGlyThrLeuLysGlu                                 110115120                                                                     TyrGluSerCysHisGluGlyLeuCysProPheLeuLeuThrGlu                                 125130135                                                                     CysProAlaCysLysGlyLeuValArgLeuSerGluLysGluHis                                 140145150                                                                     HisThrGluGlnGluCysProLysArgSerLeuSerCysGlnHis                                 155160165                                                                     CysArgAlaProCysSerHisValAspLeuGluValHisTyrGlu                                 170175180                                                                     ValCysProLysPheProLeuThrCysAspGlyCysGlyLysLys                                 185190195                                                                     LysIleProArgGluThrPheGlnAspHisValArgAlaCysSer                                 200205210                                                                     LysCysArgValLeuCysArgPheHisThrValGlyCysSerGlu                                 215220225                                                                     MetValGluThrGluAsnLeuGlnAspHisGluLeuGlnArgLeu                                 230235240                                                                     ArgGluHisLeuAlaLeuLeuLeuSerSerPheLeuGluAlaGln                                 245250255                                                                     AlaSerProGlyThrLeuAsnGlnValGlyProGluLeuLeuGln                                 260265270                                                                     ArgCysGlnIleLeuGluGlnLysIleAlaThrPheGluAsnIle                                 275280285                                                                     ValCysValLeuAsnArgGluValGluArgValAlaValThrAla                                 290295300                                                                     GluAlaCysSerArgGlnHisArgLeuAspGlnAspLysIleGlu                                 305310315                                                                     AlaLeuSerAsnLysValGlnGlnLeuGluArgSerIleGlyLeu                                 320325330                                                                     LysAspLeuAlaMetAlaAspLeuGluGlnLysValSerGluLeu                                 335340345                                                                     GluValSerThrTyrAspGlyValPheIleTrpLysIleSerAsp                                 350355360                                                                     PheThrArgLysArgGlnGluAlaValAlaGlyArgThrProAla                                 365370375                                                                     IlePheSerProAlaPheTyrThrSerArgTyrGlyTyrLysMet                                 380385390                                                                     CysLeuArgValTyrLeuAsnGlyAspGlyThrGlyArgGlyThr                                 395400405                                                                     HisLeuSerLeuPhePheValValMetLysGlyProAsnAspAla                                 410415420                                                                     LeuLeuGlnTrpProPheAsnGlnLysValThrLeuMetLeuLeu                                 425430435                                                                     AspHisAsnAsnArgGluHisValIleAspAlaPheArgProAsp                                 440445450                                                                     ValThrSerSerSerPheGlnArgProValSerAspMetAsnIle                                 455460465                                                                     AlaSerGlyCysProLeuPheCysProValSerLysMetGluAla                                 470475480                                                                     LysAsnSerTyrValArgAspAspAlaIlePheIleLysAlaIle                                 485490495                                                                     ValAspLeuThrGlyLeu                                                            500501                                                                        (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 44 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       AspLeuLeuCysProIleCysMetGlnIleIleLysAspAlaPhe                                 151015                                                                        LeuThrAlaCysGlyHisSerPheCysTyrMetCysIleIleThr                                 202530                                                                        HisLeuArgAsnLysSerAspCysProCysCysSerGlnHis                                    354044                                                                        (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 47 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GluLeuSerCysSerIleCysLeuGluProPheLysGluProVal                                 151015                                                                        ThrThrProCysGlyHisAsnPheCysGlySerCysLeuAsnGlu                                 202530                                                                        ThrTrpAlaValGlnGlySerProTyrLeuCysProGlnCysArg                                 354045                                                                        AlaVal                                                                        47                                                                            (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 44 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       LeuLeuArgCysHisIleCysLysAspPheLeuLysValProVal                                 151015                                                                        LeuThrProCysGlyHisThrPheCysSerLeuCysIleArgThr                                 202530                                                                        HisLeuAsnAsnGlnProAsnCysProLeuCysLeuPheGlu                                    354044                                                                        (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 44 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       AlaPheArgCysHisValCysLysAspPheTyrAspSerProMet                                 151015                                                                        LeuThrSerCysAsnHisThrPheCysSerLeuCysIleArgArg                                 202530                                                                        CysLeuSerValAspSerLysCysProLeuCysArgAlaThr                                    354044                                                                        (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       SerIleSerCysGlnIleCysGluHisIleLeuAlaAspProVal                                 151015                                                                        GluThrAsnCysLysHisValPheCysArgValCysIleLeuArg                                 202530                                                                        CysLeuLysValMetGlySerTyrCysProSerCysArgTyrPro                                 354045                                                                        (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      GluValThrCysProIleCysLeuAspProPheValGluProVal                                 151015                                                                        SerIleGluCysGlyHisSerPheCysGlnGluCysIleSerGln                                 202530                                                                        ValGlyLysGlyGlyGlySerValCysAlaValCysArgGlnArg                                 354045                                                                        (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 46 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      GluLeuMetCysProIleCysLeuAspMetLeuLysAsnThrMet                                 151015                                                                        ThrThrLysGluCysLeuHisArgPheCysSerAspCysIleVal                                 202530                                                                        ThrAlaLeuArgSerGlyAsnLysGluCysProThrCysArgLys                                 354045                                                                        Lys                                                                           46                                                                            (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 50 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      GluValThrCysProIleCysLeuGluLeuLeuLysGluProVal                                 151015                                                                        SerAlaAspCysAsnHisSerPheCysArgAlaCysIleThrLeu                                 202530                                                                        AsnTyrGluSerAsnArgAsnThrAspGlyLysGlyAsnCysPro                                 354045                                                                        ValCysArgValPro                                                               50                                                                            (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 47 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      GluThrThrCysProValCysLeuGlnTyrPheAlaGluProMet                                 151015                                                                        MetLeuAspCysGlyHisAsnIleCysCysAlaCysLeuAlaArg                                 202530                                                                        CysTrpGlyThrAlaGluThrAsnValSerCysProGlnCysArg                                 354045                                                                        GluThr                                                                        47                                                                            (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 48 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      PheGlnLeuCysLysIleCysAlaGluAsnAspLysAspValLys                                 151015                                                                        IleGluProCysGlyHisLeuMetCysThrSerCysLeuThrSer                                 202530                                                                        TrpGlnGluSerGluGlyGlnGlySerSerGlyCysProPheCys                                 354045                                                                        ArgCysGlu                                                                     48                                                                            (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      GlyGlyPheLysLeuValThrCysAspPheCysLysArgAspAsp                                 151015                                                                        IleLysLysLysGluLeuGluThrHisTyrLysThrCys                                       202528                                                                        (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      GlnAspLeuAlaValCysAspValCysAsnArgLysPheArgHis                                 151015                                                                        LysAspTyrLeuArgAspHisGlnLysThrHis                                             202526                                                                        (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      ThrGlyLysTyrProPheIleCysSerGluCysGlyLysSerPhe                                 151015                                                                        MetAspLysArgTyrLeuLysIleHisSerAsnValHis                                       202528                                                                        (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      ThrGlyGluLysProTyrThrCysThrValCysGlyLysLysPhe                                 151015                                                                        IleAspArgSerSerValValLysHisSerArgThrHis                                       202528                                                                        (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      ArgLysLysPheProHisIleCysGlyGluCysGlyLysGlyPhe                                 151015                                                                        ArgHisProSerAlaLeuLysLysHisIleArgValHis                                       202528                                                                        (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      SerGluGluLysProPheGluCysGluGluCysGlyLysLysPhe                                 151015                                                                        ArgThrAlaArgHisLeuValLysHisGlnArgIleHis                                       202528                                                                        (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      ProAsnGluGlnMetAlaGlnCysProIleCysGlnGlnPheTyr                                 151015                                                                        ProLeuLysAlaLeuGluLysThrHisLeuAspGluCys                                       202528                                                                        (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      ProAspAspGlyLeuValAlaCysProIleCysLeuThrArgMet                                 151015                                                                        LysGluGlnGlnValAspArgHisLeuAspThrSerCys                                       202528                                                                        (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      CCTTGTGCCTGCAGAGAGAAG21                                                       (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      CTAGGTTAACTTTCGGTGCTCCCCAGCAGGGTCTC35                                         (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      CTAGGTTAACTGGAGAAGGGGACCTGCTCGTCCTT35                                         (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      CTAGGTTAACTGCTGGCTTGGGAGGAGCACTGTGA35                                         (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      CTAGGTTAACTGCTCCCGGTGCTGGCCCGGGCCTC35                                         (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      CTAGGTTAACTGCACTGGCCGAGCTCTCCAGGGA34                                          (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      GTGATGAGAATTCAT15                                                             (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      CGATGAATTCTCATCACTGCA21                                                       (2) INFORMATION FOR SEQ ID NO:31:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      GATCGGATCCAAAAAGAAGCCCTTGTGCCTGCA33                                           (2) INFORMATION FOR SEQ ID NO:32:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                      GCCTGGTTAACTGGGC16                                                            (2) INFORMATION FOR SEQ ID NO:33:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                      GCNCCNATGGCNYTNGARC19                                                         (2) INFORMATION FOR SEQ ID NO:34:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                      GCNCCNATGGCNYTNGARA19                                                         (2) INFORMATION FOR SEQ ID NO:35:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                      GCNCCNATGGCNYTNGARG19                                                         (2) INFORMATION FOR SEQ ID NO:36:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                      GYTCNARNGCCATNGGNGC19                                                         (2) INFORMATION FOR SEQ ID NO:37:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                      TYTCNARNGCCATNGGNGC19                                                         (2) INFORMATION FOR SEQ ID NO:38:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                      CYTCNARNGCCATNGGNGC19                                                         (2) INFORMATION FOR SEQ ID NO:39:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                      AARCAYGCNTAYGTNAA17                                                           (2) INFORMATION FOR SEQ ID NO:40:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                      TTNACRTANGCRTGYTT17                                                           (2) INFORMATION FOR SEQ ID NO:41:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                      AlaProMetAlaLeuGluArg                                                         157                                                                           (2) INFORMATION FOR SEQ ID NO:42:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                      LysHisAlaTyrValLys                                                            156                                                                           (2) INFORMATION FOR SEQ ID NO:43:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                      ProGlySerAsnLeuGlySer                                                         157                                                                           (2) INFORMATION FOR SEQ ID NO:44:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                      LysAspAspThrMetPheLeuLys                                                      158                                                                           (2) INFORMATION FOR SEQ ID NO:45:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                      TCGATCGTCGACCAAAAAGAAGCCCTCCTGCCTACAA37                                       (2) INFORMATION FOR SEQ ID NO:46:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                      CTAGAGATCTCAGGGGTCAGGCCACTTT28                                                (2) INFORMATION FOR SEQ ID NO:47:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                                      CTAGAGATCTGTTAACTTTCGGTGCTCCCCAGCAGGGTCTC41                                   (2) INFORMATION FOR SEQ ID NO:48:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                      CTAGAGATCTGTTAACTGGAGAAGGGGACCTGCTCGTCCTT41                                   (2) INFORMATION FOR SEQ ID NO:49:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                                      CTAGAGATCTGTTAACTGCTGGCTTGGGAGGAGCACTGTGA41                                   (2) INFORMATION FOR SEQ ID NO:50:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                                      TCGATCGTCGACCAAAAAGAAGCCCTCCTGCCTACAA37                                       (2) INFORMATION FOR SEQ ID NO:51:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                                      CTAGAGATCTCAGGGGTCAGGCCACTTT28                                                (2) INFORMATION FOR SEQ ID NO:52:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                                      TCGATCGTCGACCGCCTCCAGCTCAGCCCCTGAT34                                          (2) INFORMATION FOR SEQ ID NO:53:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                                      GATCGGATCCGGAGACACAGATTCCAGCCCC31                                             (2) INFORMATION FOR SEQ ID NO:54:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:                                      GATCGAATTCTTAACTCTTCGGTGCTCCCCAGCAG35                                         (2) INFORMATION FOR SEQ ID NO:55:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:                                      GATCGGATCCTTGTGGTGTGTGGGGGTTGT30                                              (2) INFORMATION FOR SEQ ID NO:56:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:                                      CCTGGCTGGCCTAATGT17                                                           (2) INFORMATION FOR SEQ ID NO:57:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 60 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:                                      GATCGACTCGAGATGCCCAAGAAGAAGCGGAAGGTGGCTGCAGCCAGTGT50                          GACTTCCCCT60                                                                  (2) INFORMATION FOR SEQ ID NO:58:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:                                      CTCTGGCGAAGAAGTCC17                                                           (2) INFORMATION FOR SEQ ID NO:59:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:                                      GATCGGATCCGCCTCCAGCTCAGCCCCTGAT31                                             (2) INFORMATION FOR SEQ ID NO:60:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:                                      GATCGGATCCAGCCAGCAGCTTCTCCTTCAC31                                             (2) INFORMATION FOR SEQ ID NO:61:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:                                      GATCGGATCCTTGTGGTGTGTGGGGGTTGT30                                              (2) INFORMATION FOR SEQ ID NO:62:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:                                      GATCGGATCCGCTCAGGCTCTTTTGGGGCA30                                              __________________________________________________________________________

We claim:
 1. An isolated TRAF selected from the group consisting ofTRAF1 having the amino acid sequence depicted in SEQ. ID. NO:2 and TRAF2having the amino acid sequence depicted in SEQ. ID. NO:4.